thermo scientific pierce protein purification …...thermo scientific pierce protein purification...

Aga

rose

Bea

d O NH

Bead

NH2NH2

H2N

H2N+

O

O

O

O

O

N

Thermo Scientific Pierce Protein Purification Technical Handbook

Version 2

Introduction for Protein Purification 1-5

Purification Accessories – Protein Extraction, Binding and Elution Buffers 6-11

Protease and Phosphatase Inhibitors 8 for Protein Purification Buffers for Protein Purification 9Spin Cups and Columns 10Disposable Plastic and Centrifuge Columns 11

Fusion Protein Purification 12-21

His-Tagged Protein Purification Resin 14-15Cobalt Resin, Spin Columns and 16-17 Chromatography Cartridges High-quality purification of GST-fusion proteins 18-19GST- and PolyHis-Tagged Pull-Down Assay Kits 20-21

Covalent Coupling of Affinity Ligands to Chromatography Supports 22-35

Covalent Immobilization of Ligands 22-25Products for Immobilizing Ligands 26-29 through Primary Amines Products for Immobilizing Ligands 30-32 through Sulfhydryl Groups Products for Immobilizing Ligands 33 through Carbonyl Groups Products for Immobilizing Ligands 34-35 through Carboxyl Groups

IP/Co-IP 36-45

Immunoprecipitation 36 Traditional Methods vs. 37 Thermo Scientific Pierce Innovations for Co-IP Approaches to Co-IP Free of Antibody Interference 37Optimization Parameters in IP and Co-IP 38Evaluating a Co-IP-Captured Interaction 38IP and Co-IP Kits 39-45

Protein Enrichment 46-57

Phosphoprotein Enrichment Kits 46-47 SH2 Domain Phosphotyrosine Capture Kits 48-50 Use of Titanium Dioxide for 51-52 Phosphopeptide Enrichment Pierce Fe-NTA Phosphopeptide Enrichment Kit 53-54Cell Surface Protein Isolation Kit 55Glycoprotein Isolation Kits 56Ubiquitin Enrichment Kit 57

Antibody Purification 58-67

Overview 58 Immobilized Protein L, Protein A, Protein G 59-61 and Protein A/G IgG Binding and Elution Buffers for 62 Protein A, G, A/G and L Melon Gel Purification Products 63Thiophilic Gel Antibody Purification 64IgM and IgA Purification 65

Avidin:Biotin Binding 66-69

Biotin-binding Proteins 66Immobilized Avidin Products 67Immobilized Streptavidin Products 67Immobilized NeutrAvidin Products 68Immobilized Monomeric Avidin and Kit 68Immobilized Iminobiotin and Biotin 69

FPLC Cartridges 70-77

Overview 70His-tagged Protein FPLC Purification 71-72GST-Tagged Protein FPLC Purification 72Antibody FPLC Purification 73-75Phosphoprotein FPLC Purification 75Biotinylated Protein FPLC Purification 76Protein Desalting 77

Affinity Supports 78-80

Table of Contents

To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1


General Protein Purification Techniques

Protein purification is essential for a host of biochemical appli-cations. However, with thousands of proteins each displaying unique characteristics, it is important to develop a strategy for purification that delivers the correct yield, purity and activity needed for downstream applications. Forlowresolution/highyieldproteinpurification,methodssuchasfractionalprecipitationusingsaltssuchasammoniumsulfateexist.Forapplicationsrequiringthehighestpurityandrelativelysmallamountsofprotein,affinitypurificationtechniquescanbechosentoselectivelyextractatargetproteinfromthecomplexmixtureofproteinsfoundincellortissueextracts.Table1summarizesafewgeneralstrategies.

Eachoftheseproteinpurificationtechniquesrequiresspecificbuffers(mobilephase),chromatographyresins(solidphase)andcolumnaccessories.Thesethreecomponentscanbeusedinavarietyofdifferentconfigurations,basedonthescaleofpurifica-tionandavailableequipment.Commonformatsinclude:

Batch purification Mixingthemobileandstationaryphasesinaconicaltubeandseparatingthetwoviacentrifugation.Batchmethodpurificationcanbeperformedatanyscale.However,itismostcommonlyreservedformicrocentrifugetubescalepurificationsinvolving10-200μLofresin.Inbatchmethodpurification,washandelutionfractionsareseparatedfromtheresinaftercentrifugingtopellettheresinbeads.Theliquidcannotberemovedcompletelybecausesomeofitiscontainedwithinthevolumeofporousbeadpellet.Consequently,aportionofeachfractionaboutequaltothevolumeofresinusedisleftbehindinthepellet,makingwashesandelutionsomewhatinefficient.

Gravity flow chromatography Passivelyaddingthemobilephasetopackedcolumns,withoutmechanicallyincreasingtheflowrate.Gravityflowcom-monlyuses1mL-to5mL-packedcolumnsset-uponabenchorinachromatographyrefrigerator.Largercolumnscanbepackedtosupportlargerscaleproteinpurification.However,theweightoftheresininthecolumnmustbeconsideredtopreventdamagetoresinatthebottomofthecolumn.Gravityflowallowsforextendedbinding,washingandelutiontimes,whichisidealforsampleswithlowbindingaffinity.

Introduction to Protein Purification

Table 1. Summary of protein purification techniques.

Resolution/ purity Technique Protein Yield Description

Low

Ammoniumsulfateprecipitation High Fractionalprecipitationofproteinsbasedontheirsolubilityinsaltsolutionsofvaryingsaturation

Hydrophobicinteractionchromatography High Separationofproteinsbasedintheirsurfacehydrophobicity

Sizeexclusionchromatography(SEC) Medium Separationofproteinsbasedonmolecularsize

Medium

Ionexchangechromatography Medium SeparationbasedonproteinchargeataparticularpH

Gelelectrophoresis Medium Two-stepseparationofproteinbasedonsizeusingpolyacrylimidegelelectrophoresis(SDS-PAGE)andchargeusingisoelectricfocusing.Note:Thisisadenaturingmethodthatresultsinacompletelossofproteinactivity.

Proteomefractionation Medium Enrichmentofclassesofproteins,suchas:•phosphoproteinsusingimmobilizedmetalaffinitychromatography(IMAC)•glycoproteinsusingimmobilizedlectins•nitrosylatedorpalmitylatedproteinsusingthebiotinswitchassay•organelle-specificproteomesusingcellularfractionationtechniques

High

Affinitypurification Low Selectivepurificationusingaffinitytagsattachedtothetargetgene,suchaspolyhistidine(6xHis),glutathioneS-transferase(GST)ormaltosebindingprotein(MBP)

Immunoprecipitation Lowest Antibody-basedextractionofasingleproteinspecies,typicallyfromcellortissuelysatesh

2 For more information, or to download product instructions, visit www.thermoscientific.com/pierce

Spin cup purificationSeparatingthemobileandstationaryphasesusingcentrifugationinpackedspintubeswithfiltersthatretainresinincolumn.Spinpurificationcanalsobeperformedwithspinplates,whereeachwellofa96-wellmicrotiterplatehasafilterbaseandispackedwiththeappropriatechromotagraphyresin.Thespincuppurificationmethodprovidesimprovedefficiencyofwashandelutionstepscomparedtothebatchmethod.Centrifugationseparatestheliquidfractionbypullingitthoroughlyfromtheresin,whichisretainedwithinthespincupapparatus.Spincuppurifica-tionismostappropriatewhen50-300μLofimmobilizedligandresinisused.

Magnetic purificationIsolatingthestationaryphaseusingmag-nets.Magneticbeadsarecommonlyironoxideparticleswhicharecoatedwithaligandtopurifyaproteintarget.Anexampleofthisisamagneticparticlecoatedwithreducedglutathione(GSH)usedtopurifyGST-taggedrecombinantproteins(Product#88821).Advantagesofusingmagneticbeadsincludeeasyhandling,minimallossofresinfrompipettingandcompatibilitywithhighthroughoutautomatedsystemssuchastheThermoScientificKingFisher96instrument.

Fast protein liquid chromotagraphy (FPLC)Usingchromatographycartridgespre-packedwiththestationaryphaseandaseriesofpumpsandUVdetectorstomoveandmonitorthemobilephase.TheadvantagesofusingFPLCincludereducedpurificationtimesandtheabilitytoimproveresolutionbylinkingmultiplecolumnsintandemforgreaterseparation.

Affinity Purification

Variousmethodsareusedtoenrichorpurifyatargetproteinfromotherproteinsandcomponentsinacrudecelllysateorothersample.Themostpowerfulofthesemethodsisaffinitypurification,alsocalledaffinitychromatography,wherebytheproteinofinterestispurifiedusingitsspecificbindingpropertiestoanimmobilizedligand.

Affinitypurificationmakesuseofspecificbindingthatoccursbetweenmoleculesandisusedextensivelyfortheisolationofbiologicalmolecules.Asinglepassthroughanaffinitycolumncanachievea1,000-to10,000-foldpurificationofaligandfromacrudemixture.Fromasingleaffinitypurificationstep,itispossibletoisolateacompoundinaformpureenoughtoobtainasinglebanduponSDS-PAGEanalysis.Weofferanumberofimmobilizedproteinorligandproductsforaffinitypurificationofantibodies,fusion-taggedproteins,biotinylatedproteinsandotherproteinsforwhichanaffinityligandisavailable.

Inaffinitypurification,aligandisimmobilizedtoasolidsupport.Onceimmobilized,itspecificallybindsitspartnerundermildbufferconditions(oftenphysiologicalconditionssuchasphosphatebufferedsaline).Afterbindingtothepartnermolecule,thesupportiswashedwithadditionalbuffertoremoveunboundcomponentsofthesample.Anelutionbufferisadded,disruptingtheinteractionbetweentheligandanditsbindingpartnerbypHextremes(loworhigh),highsalt,detergents,chaotrophicagentsortheremovalofsomefactorrequiredforthepairtobind.Oncereleased,thebindingpartnercanberecoveredfromthesupportusingadditionalelutionbuffer.Theelutionbuffercanthenbeexchangedbydialysisordesaltingintoamoresuitablebufferforstorageordownstreamanalysis.

Activatedaffinitysupportproductsandkitsenablearesearchertoimmobilizenearlyanytypeofligandtopurifyitsbindingpartner(s).Forexample,ifapeptideantigenisusedtoimmunizeanimalsandproduceantibodies,thesamepeptidecanbeimmobilizedtoagelsupportandusedtoaffinity-purifythespecificantibodyfromanimalserum.Alternatively,ifaspecificantibodyisavailableagainstaparticularproteinofinterest,itcanbeimmobilizedtoasupportandusedtoaffinity-purifytheproteinfromcrudecelllysate.Purificationwithrespecttonearlyanybindinginteractioncanbemadebythisapproach.


3To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.

Affinitypurificationproductsusingeitherimmobilizedligandsoractivatedaffinitysupportchemistriesareavailableforuseinseveraldifferentformats.Mostcommonly,porousbeadedgelsupportsareusedforgravity-column,spin-columnorbatch-scalepurificationprocedures.Coatedmicroplatesareavailableforhigh-throughputscreeningapplications,andmagneticparticlesareespeciallyusefulforautomatedproteinpurification.

Proteinsandothermacromoleculesofinterestcanbepurifiedfromcrudeextractsorothercomplexmixturesusingavarietyofmethods.Precipitationisperhapsthesimplestmethodforseparatingonetypeofmacromoleculefromanother.Forexample,nucleicacidscanbeprecipitatedandtherebypurifiedfromundesiredmoleculesinsolutionusingethanol,andproteinscanbeselectivelyprecipitatedinthepresenceofammoniumsulfate.

Mostpurificationmethodsinvolvesomeformofchromatographywherebymoleculesinsolution(mobilephase)areseparatedbasedondifferencesinchemicalorphysicalinteractionwithastation-arymaterial(solidphase).Gelfiltration(alsocalleddesalting,sizeexclusionchromatographyorSEC)usesaporousgelmaterialtoseparatemoleculesbasedonsize;largemoleculesareexcludedfromtheinternalspacesofthegelmaterialwhilesmallmoleculesentertheresinpores,resultinginalongerpaththroughthecol-umn.Inionexchangechromatography,moleculesareseparatedaccordingtothestrengthoftheiroverallionicinteractionwithasolid-phasematerial.Bymanipulatingbufferconditions,moleculesofgreaterorlesserioniccharactercanbeboundtoordissociatedfromthesolid-phasematerial.

Incontrast,affinitychromatographyoraffinitypurificationmakesuseofspecificbindinginteractionsbetweenmolecules.Aparticularligandischemicallyimmobilizedor“coupled”toasolidsupportsothatwhenacomplexmixtureispassedoverthecolumn,onlythosemoleculeshavingspecificbindingaffinitytotheligandarepurified.Affinitypurificationgenerallyinvolvesthefollowingsteps:

1.Incubatecrudesamplewiththeimmobilizedligandsupportmaterialtoallowthetargetmoleculeinthesampletobindtotheimmobilizedligand.

2.Washawayunboundsamplecomponentsfromsolidsupport.

3.Elute(dissociateandrecover)thetargetmoleculefromtheimmobilizedligandbyalteringthebufferconditionssothatthebindinginteractionnolongeroccurs.

Ligandsthatbindtogeneralclassesofproteins(e.g.,ProteinAforantibodies)orcommonlyusedfusionproteintags(e.g.,glutathioneforGST-taggedproteins)areavailableinpre-immobilizedformsreadytouseforaffinitypurification.Alternatively,morespecializedligandssuchasspecificantibodiesorantigensofinterestcanbeimmobilizedusingoneofseveralactivatedaffinitysupports;forexample,apeptideantigencanbeimmobilizedtoasupportandusedtopurifyantibodiesthatrecognizethepeptide.

Mostcommonly,ligandsareimmobilizedor“coupled”directlytosolidsupportmaterialbyformationofcovalentchemicalbondsbetweenparticularfunctionalgroupsontheligand(e.g.,primaryamines,sulfhydryls,carboxylicacids,aldehydes)andreactivegroupsonthesupport.However,othercouplingapproachesarealsopossible.IntheThermoScientificGSTOrientationKit(Product#78201),forexample,aGST-taggedfusionproteinisfirstboundtoanimmobilizedglutathionesupportbyaffinityinteractionwiththeGSTtagandthenchemicallycrosslinkedtothesupport.TheimmobilizedGST-taggedfusionproteincanthenbeusedtoaffinity-purifyitsbindingpartner(s).Likewise,theThermoScientificPierceCrosslinkImmunoprecipitationKits(Product#26147)andThermoScientificIgGOrientationKits(Product#44990)involvebindingandsubsequentcrosslinkingofanantibodytoimmobilizedProteinA,A/G.

Historically,researchershaveusedaffinitypurificationprimarilytopurifyindividualmoleculesofinterest.Increasingly,proteomicsresearchfocusesondeterminationofdiseasestates,celldifferentiation,normalphysiologicalfunctionsanddrugdiscoveryinvolvinginteractionandexpressionofmultiplemoleculesratherthanindividualtargets.Consequently,theuseofaffinitymethodshasexpandedtopurificationofnativemolecularcomplexesandformsthebasisforco-immunoprecipitation(co-IP)and“pull-down”assaysinvolvingprotein:proteininteractions.

1. Immobilize the antigen to an appropriate support.

4. Bind anti-antigen antibodies.

5. Wash off unbound antibodies.

6. Elute anti-antigen antibodies.

2. Quench the unreacted sites and wash.

3. Add the antibody solution.

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

3ml

2ml

1ml

5ml

4ml

5ml

4ml

5ml

4ml

Antigen

Antigen

Antigen

Antigen

Antigen

TBS

Typical antibody purification using an immobilized antigen column.


Affinity Purification Supports

Affinitypurificationinvolvestheseparationofmoleculesinsolution(mobilephase)basedondifferencesinbindinginteractionwithaligandthatisimmobilizedtoastationarymaterial(solidphase).Thesolidphaseinaffinitypurificationisasupportormatrixmaterialthatabiospecificligandmaybecovalentlyattached.Typically,thematerialtobeusedasanaffinitymatrixisinsolubleinthesysteminwhichthetargetmoleculeisfound.Usually,butnotalways,theinsolublematrixisasolid.Hundredsofsubstanceshavebeendescribedandemployedasaffinitymatrices.

Usefulaffinitysupportsarethosewithahighsurfaceareatovolumeratio,chemicalgroupsthatareeasilymodifiedforcovalentattachmentofligands,minimalnonspecificbindingproperties,goodflowcharacteristics,andmechanicalandchemicalstability.Whenchoosinganaffinitysupportormatrixforanyseparation,themostimportantquestiontoansweriswhetherareliablecommercialsourceexistsforthedesiredmatrixmaterialinthequantitiesrequired.Fortunately,weofferawiderangeofpracticalandefficientmatricesinvolumesrangingfrom1mLtomuchlargerbulkquantities.

Porous Beaded ResinsPorousbeadedsupportsgenerallyprovidethemostusefulproper-tiesforaffinitypurificationofproteins.Weofferaffinitypurifica-tionproductsintwomainporousgelsupportformats:crosslinkedbeadedagaroseandThermoScientificUltraLinkBiosupport.Thevariousfeaturesofthesetwosupportsarelistedintheaccompanyingtable.Agaroseisgoodforroutineapplicationsbutcrusheseasily,makingitsuitableforgravity-flowcolumnorsmall-scalebatchproceduresusinglow-speedcentrifugation.UltraLinkBiosupportisincompressibleandcanbeusedinhigh-pressureapplicationswithaperistalticpumporotherliquidchromatogra-physystem.Inaddition,UltraLink®Supportsdisplayextremelylownonspecificbindingcharacteristicsbecausetheyarepolyacryl-amide-based.Bothsupportsperformwellintypicalgravity-flowandspincolumnpurificationandimmunoprecipitationprocedures.

Magnetic ParticlesWhenamatrixisrequiredforaffinitypurificationofcellswithinapopulation,werecommendThermoScientificMagnaBindBeads.Magneticaffinityseparationisaconvenientmethodforisolatingantibodies,antigens,lectins,enzymes,nucleicacidsandcellswhileretainingbiologicalactivity.Samplescontainingthemole-culeofinterestareincubatedwithbeadsthatarederivatizedwithanantibodyorotherbindingpartner.ArareearthmagnetisusedtopulltheMagnaBind™Beadsoutofsolutionandontoasurface.Thebuffercanbecarefullyremoved,containinganynon-boundmoleculesorcells.

MagnaBindBeadsconsistofasilanizedsurfaceoveranironoxidecore(seeTable2).Thesilanizedsurfacehasbeenderiva-tizedtocontainactivegroups,suchascarboxylicacidsorprimaryamines,orspecificaffinitymoleculessuchasstreptavidin;ProteinA;ProteinG;orgoatanti-mouse,anti-rabbitoranti-ratIgG.DuetothenatureoftheMagnaBindBeads,strongelutionconditionsarenotrecommendedwiththeseproducts.Seepage80foracompletelistingofMagnaBindSupports.



Table 2. Characteristics of underivatized Thermo Scientific MagnaBind Beads.

Composition Silanizedironoxide

Magnetization 25–35EMU/g

Type of Magnetization Superparamagnetic(nomagneticmemory)

Surface Area >100m2/g

Settling Rate 4%in30minutes

Effective Density 2.5g/mL

Number of Beads 1x108beads/mg

pH Stability Aqueoussolution,abovepH4.0

Concentration 5mg/mL

Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with antibiotic medium or g-irradiated.

MicroplatesPolystyrenemicroplatesareanothertypeofmatrixcommonlyusedforimmobilizationofproteins.Proteinspassivelyadsorbtothepolystyrenesurfacethroughhydrophobicinteractions.Generally,thisadsorptionofproteinsontothepolystyrenesurfaceoccursbestincarbonate/bicarbonatebufferatanalkalinepH(9.0–9.5).Inaddition,polystyrenesurfacescanbederivatizedwithcertainchemistriesthatwillallowpeptidesandothernonproteinmoleculestoadheretothesurfacetoperformaffinityassaysinthewellsoftheplates.

Weofferprecoatedplatestoallowresearchersaneasy-to-use,consistentmethodforaffinitypurificationoridentificationofspecificmoleculesofinterest.Theplatesofferedincludethosespecificforfusionproteins(6xHis,GSTandGFP),antibodies(ProteinA,ProteinG,ProteinA/G,ProteinL,goatanti-mouseandgoatanti-rabbitIgG),biotin(streptavidinandThermoScientificNeutrAvidinProtein)andthosewithreactivechemistries(maleicanhydrideandmaleimide)toallowbindingofnonproteinsamplesthatdonotadsorbtotheplasticmicroplatewellsurface.Onlyselectedmicroplateproductsarefeaturedinthishandbook.Foracompleteselectionofprecoatedplates,visitwww.thermosci-entific.com/pierce.

Thereareavarietyofactivatedsupportsthatallowaresearchertopurifyproteinsandotherbiologicalmoleculesofinteresteitheraloneorwhenpresentincomplexeswiththeirbindingpartners.Manyofthesesupportsarediscussedonthefollowingpages.

Physical properties of porous gel supports.

Support

4% Agarose (crosslinked beaded agarose)

6% Agarose(crosslinked beaded agarose)

Thermo Scientific UltraLink Biosupport (co-polymer of crosslinked bis-acrylamide and azlactone)

Bead 45–165μm 45–165μm 50–80μm

Exclusion Limit 20,000,000daltons 4,000,000daltons 2,000,000daltons(1,000Åporesize)

Durability Crushesunderpressure Crushesunderpressure Sturdy(>100psi,6.9bar)*

Types of Chromatography Gravityandsmallspincolumns Gravityandsmallspincolumns FPLCSystems,mediumpressure,gravityflow

Coupling Capacity Medium Medium High

pH range 3–11 3–11 3–11

Form Preswollen Preswollen DryorPreswollen

* Note: The indicated maximum pressure of 100 psi refers to the maximum pressure drop across the gel bed that the support can withstand. It does not necessarily refer to the indicated system pressure shown on a liquid chromatography apparatus because the system pressure may not actually be measuring the pressure drop across the column. Typical system pressures are usually much higher due to

pumping through small I.D. tubing, auto-samplers, detectors, etc. When packed into a 3mm ID x 14cm height glass column, these exclusive supports have been run to approximately 650 psi (system pressure) with no visual compression of the gel or adverse effects on chromatography. These columns can be run at linear flow rates or 85–3,000cm/hour with excellent separation characteristics.


Protein Extraction, Binding and Elution Buffers

Protein purification is preceeded by expression of the target protein in a host organism. In in vitro protein expression, the simple introduction of DNA and a bacteriophage RNA polymerase to a cell free lysate initiates protein production, with no extrac-tion reagents required before purification. For in vivo protein expression, such as in E. coli or tissue culture cells, expression can be driven from constituitive promoters or it can be induced chemically during bacterial growth or induced genetically through transient DNA transfection into tissue culture cells. For in vivo protein expression, the total protein content of a cell culture or tissue sample must be extracted from the cell's mem-branes and organelles before a chromatography resin can be used for purification. A list of Thermo Scientific Pierce Protein Extraction Reagents for different cell and tissue type is listed on page 7. Additionally, protease and phosphatase inhibitor cocktails that can be used to preserve protein integrity during extraction (see page 8).

Mostaffinitypurificationproceduresinvolvingprotein:ligandinter-actionsusebindingbuffers,suchasphosphatebufferedsaline(PBS),atphysiologicpHandionicstrength.Thisisespeciallytruewhenantibody:antigenornativeprotein:proteininteractionsarethebasisfortheaffinitypurification.Oncethebindinginteractionoccurs,thesupportiswashedwithadditionalbuffertoremoveunboundcomponentsofthesample.

Nonspecific(e.g.,simpleionic)bindinginteractionscanbemini-mizedbymoderateadjustmentstosaltconcentrationorbyaddinglowlevelsofdetergentinthebindingand/orwashbuffer.Finally,elutionbufferisaddedtobreakthebindinginteractionandreleasethetargetmolecule,whichisthencollectedinitspurifiedform.ElutionbuffercandissociatebindingpartnersbyextremesofpH(loworhigh),highsalt(ionicstrength),theuseofdetergentsorchaotropicagentsthatdenatureoneorbothofthemolecules,removalofabindingfactor,orcompetitionwithacounterligand.Inmostcases,subsequentdialysisordesaltingisrequiredtoexchangethepurifiedproteinfromelutionbufferintoamoresuitablebufferforstorageordownstreamanalysis.Formoreinformationondialysisordesalting,downloadorrequestourafreehigh-performancedialysistechnicalhandbook.

Themostwidelyusedelutionbufferforaffinitypurificationofproteinsis0.1Mglycine•HCl,pH2.5–3.0.Thisbuffereffectivelydissociatesmostprotein:proteinandantibody:antigenbindinginteractionswithoutpermanentlyaffectingproteinstructure.However,someantibodiesandproteinsaredamagedbylowpH,soelutedproteinfraction(s)shouldbeneutralizedimmediatelybycollectingthefractionsintubescontaining1/10thvolumeofalkalinebuffersuchas1MTris•HCl,pH8.5.Otherelutionbuffersforaffinitypurificationofproteinsarelistedintheaccompanyingtable.Inaddition,weofferseveralpreformulatedbindingandelutionbuffersdesignedforaffinitypurificationinvolvingantibodies.

Common elution systems for protein affinity purification.

Condition Buffer

pH 100mmglycine•HCl,pH2.5–3.0100mmcitricacid,pH3.050–100mmtriethylamineortriethanolamine,pH11.5150mmammoniumhydroxide,pH10.5

Ionicstrengthand/orchaotropiceffects

3.5–4.0Mmagnesiumchloride,pH7.0in10mmTris5Mlithiumchloridein10mmphosphatebuffer,pH7.22.5Msodiumiodide,pH7.50.2–3.0sodiumthiocyanate

Denaturing 2–6Mguanidine•HCl2–8Murea1%deoxycholate1%SDS

Organic 10%dioxane50%ethyleneglycol,pH8–11.5(alsochaotropic)

Competitor >0.1Mcounterligandoranalog

Protein Purification Accessories

Cell Lysis Technical Handbook

This50-pagehandbookprovidesprotocolsandtechnicalandproductinformationtohelpmaximizeresultsforprotein/geneexpressionstudies.Thehandbookprovideshelpfulhintsandtroubleshootingforcelllysis,proteinpurification,cellfractionation,proteaseinhibitorsandproteinrefolding.(#1601756)


Thermo Scientific Pierce Protein Extraction Reagents.

Name Description Organisms/Samples

B-PER® - Bacterial Protein Extraction Reagent Product # 90084

Efficient,gentlelysisandextractionofsolubleproteinsfromE. coliandotherbacterialcells.Usesmildnonionicdetergentstodisruptcellsandsolubilizingproteinswithoutdenaturation,eliminatingtheneedforharshmechanicalprocedureslikesonication.

Gram(-)bacteria,S. aureus,H. pylori,E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.

B-PER IIProduct # 78260

SimilartoB-PER,butoptimizedforlowcelldensity,orforproteinswithlowexpressionlevels.


B-PER PBS Product # 78266

SimilartoB-PER,butinPhosphateBuffer.Thisaminefreeformulationisidealforamine-reactivelabelingand/orcrosslinkingapplications.


B-PER with Enzymes Product # 90078

SimilartoB-PER,butkitcontainsDNAseIandlysozyme,whichimprovecellmembraneandDNAdigestionforincreasedyields,andincreasestherecoveryoflargemolecularweightproteinsandinsolubleproteinsfrominclusionbodies.

Gram(-)bacteria,S. aureus, H. pylori, E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.

B-PER Direct with Enzymes Product # 90080

SimilartoB-PERwithEnzymes,butbacteriacanbelyseddirectlyincellculturemedia;idealforscreening96-wellmicroplatesamples.

Gram(-)bacteria,S. aureus, H. pylori, E. colistrainsBL21(D3)>JM109>DH5a>M15,Archaebacteria,nematodesandAcinetobactersp.

Y-PER® - Yeast Protein Extraction Reagent Product # 78990

Easy-to-usesolutiongentlydisruptsthetoughyeastcellwallinlessthan20minutesatroomtemperature,usingamilddetergent.Nomechanicaldisruptionneeded;yieldsmorethantwiceasmuchproteinasglassbeadmethods.

S. cerevisiae,Schizo-saccharomyces pombe,C. albicans,B. subtilis,E. coli,P. pastoris,Strep. avidiniiandAcinetobactersp.

Y-PER Plus Product # 78998

MorestringentthanY-PER,butentireformulation(includingdetergent)aredialyzable.

Yeast(S. cerevisiae)andAcinetobactersp.

M-PER® - Mammalian Protein Extraction Reagent Product # 78501

Highlyefficienttotalproteinextractionfromculturedmammaliancells;extractsproteinsinnondenaturedstate,enablingproteintobedirectlyimmunoprecipitated;amine-freeandfullydialyzable;adheredcellscanbedirectlylysedinplateorafterscrapingandwashinginsuspension.

Culturedmammaliancells,COS-7,NIH3T3,Hepa1-6,293,CHO,MDA,MB231andFM2

P-PER - Plant Protein Extraction Reagent Product # 89803

Containsorganiclysingreagentandtwoaqueousreagents,which,inconjuctionwithmildmechanicalagitation,effectivelyextracthighqualityproteinextractsfromplantleaves,stem,root,seedandflowercellswithoutliquidnitrogenorharshmechanicalaids,suchasmortarandpestle.

Multipleplantorgans(leaf,stem,root,seedandflowers);multipleplantspecies(Arabidopsis,tobacco,maize,soybeans,peas,spinach,riceandotherplanttissues);andfresh,frozenanddehydratedplanttissues

T-PER® - Tissue Protein Extraction Reagent Product # 78510

Simple,easytousereagentforextractingtotalproteinfromtissuein1:20(w/v)oftissuetoT-PER,usingcentrifugationtopelletcell/tissuedebris.Milddetergentisdialyzable.

Heart,liver,kidneyandbrain.

I-PER - Insect Cell Protein Extraction Reagent Product # 89802

Optimizedmildnonioinicdetergentformulationprovidesmaximumextractionofsolubleproteinsfrominsectcells;betteryieldthansonication;canbeusedforbothsuspendedoradherentinsectcells

Baculovirus-infectedinsectcellsgrowninsuspensionormonolayerculture.

NE-PER® - Nuclear and Cystoplasmic Extraction Kit Product # 78833

Obtainfunctionalconcentratednuclearextractsandcytoplasmicfractionsfrommammaliancellsandtissuesinlessthantwohours,eliminatingtheneedforfreeze/thawcycles,Douncehomogenization,lengthycentrifugationtimesandcold-roomwork.

Tissue:calfliver.Tissue:mouseheart,kidney,lungandliver;Culturedcells:epithelial(HeLa),fibroid(COS-7),kidney(NIH3T3),liver(Hepa1)andbrain(C6).

Mem-PER® - Eukaryotic Membrane Protein Extraction Kit Product # 89826

Efficient,gentlereagentsthatsolubilizeandisolatemembraneproteinsfrommammalianandyeastcells,aswellassoftandhardtissues,inlessthananhour.Minimalcrosscontamination(lessthan10%)ofhydrophilicproteinsintothehydrophobic(membraneprotein)fraction

Culturedcells:brain(C6),epithelial(HeLa),fibroblasts(NIH3T3)andyeast(S. cerevisiae).

Subcellular Protein Fractionation Kit Product # 78840

Includesacombinationofreagentsforstepwiselysisofmammaliancellsintofunctionalcytoplasmic,membrane,solublenuclear,chromatin-bound,andcytoskeletalproteinfractionsinasinglekit;includesastabilizednucleaseandproteaseinhibitors.Extractsfromeachcompartmenthavelessthan15%contaminationbetweenfractions,withsufficientpurityforstudyingproteinlocalizationandredistribution.

Culturedmammaliancells.

Mitochondrial Isolation Kit for Cultured Cells Product # 89874

Isolateintactmitochondriafromculturedmammaliancellsinapproximately40minutes,withanoptionalDouncehomogenizationprotocolforincreasedyield.

Mammaliancells.

Mitochondrial Isolation Kit for Tissues Product # 89801

Isolateintactmitochondriafromsoftorhardtissueinlessthan60minutes,withanoptionalDouncehomogenizationprotocolforincreasedyield.


Lysosome Enrichment Kit for Tissues and Cells Product # 89839

Usesdensitygradientcentrifugationtoseparatelysozymefromcontaminatingcelluarstructuresinbothmammaliancellsandsoftandhardtissue.

Tissuesandculturedcells.

Peroxisome Enrichment Kit for Tissues Product # 89840

Usesdensitygradientcentrifugationtoseparateperoxisomefromcontaminatingcelluarstructuresinbothsoftandhardtissue.


Nuclei Enrichment Kit for Tissue Product # 89841

Usesdensitygradientcentrifugationtoseparatenucleifromcontaminatingcelluarstructuresinbothsoftandhardtissue


Pierce® RIPA BufferProduct # 89900

Extractscystoplasmic,membrane,andnuclearproteinsfromculturedmammaliancells;canbeusedforbothplatedcellsandcellspelletedfromsuspensioncultures.Proteaseandphosphataseinhibitorsarecompatiblewiththisformulation.

Culturedmammaliancellsandcytoplasmic,membraneandnuclearproteins.

Pierce IP Lysis Buffer Product # 87787

Gentlyextractscytoplasmic,membraneandnuclearproteinswhilemaintainingproteincomplexesforimmunoprecipitation(IP),Pulldowns,andco-IP;doesnotliberateDNAwhichcancausehighviscosity.

Culturedmammaliancells.

For more details on these products, request the Cell Lysis Technical Handbook (#1601756) at www.thermoscientific.com/pierce

8 For more information, or to download product instructions, visit www.www.thermoscientific.com/pierce


Protease and Phosphatase Inhibitors for Protein Purification

Thermo Scientific Halt Protease and Phosphase InhibitorsOurbroad-spectrumproteaseandphosphataseinhibitorcocktailsandindividualproteaseinhibitorsaccommodatespecificandgeneralneedsincelllysisandproteinextractionmethods.

Highlights•HaltProteaseInhibitorCocktailstargetserine,cysteineand

asparticacidproteasesandaminopeptidases.MetalloproteasesareinhibitedbytheoptionaladditionofEDTA.Individualproteaseinhibitorstargetingseparateclassesofproteasesarealsoavailableforcustomcocktaildevelopment.

•Halt™PhosphataseInhibitorCocktailscontainchemicalcom-poundsthattargetserine/threonineandtyrosinephosphatases.

•TheHaltProteaseandPhosphataseInhibitorCocktailpreventsproteindegradationandpreservesphosphorylationsimultaneously,providingprotectionthatissimilartotheindividualcocktails.

ForacompletelistingoftheHaltProteaseandPhosphataseInhibitorsatwww.thermoscientific.com.

Buffers for Protein Purification

Thermo Scientific BupH Pack Dry Blend BuffersBupH®Packsarepre-blendedandpre-measureddrymixturesofcommonlyneededbuffersthatareeasytoprepare;simplyemptycontentsoffoilenvelopepackintoabeaker,addultrapurewater,andstirtodissolve.ThepackseliminateweighingtimeandtediouspHadjustments.BupHPackDryBlendBuffersareofferedforuseincommonlaboratorytechniquesandtosupportotherPierceProteinResearchProducts.

Highlights:•Convenient–dissolvecontentsofoneenvelopein500mlofwater andthebufferisreadytouse•Save time and trouble–noweighing,nopHadjustment,noneed

tostockindividualcomponentsandnoneedtomakeandstorelargevolumesofstocksolutioninadvanceofdailyneeds

•Long shelf life–stockingandstorageasdrypackseliminates concernsaboutlong-termstabilityofstocksolutions•Eliminate variables–ourqualitycontrolensuresthateverypack willyieldthesame,consistentbuffer

Ordering Information Product #

Description

Pkg. Size

Applications

Formulation of each pack after reconstitution

U.S. Price

28384 Borate Buffer 40packs Proteinmodificationproceduresthatrequireamine-freebufferatalkalinepH

500mLof50mMborate,pH8.5 $132

28382 Carbonate-Bicarbonate Buffer 40packs MicroplateproteinandantibodycoatingforELISAorRIA

500mLof0.2Mcarbonate-bicarbonate,pH9.4 $125

28388 Citrate-Carbonate Buffer 10packs ProteinimmobilizationtoUltraLink®

Biosupport(Product#53110)100mLof0.6Msodiumcitrate,0.1Msodiumcarbonate,pH9

$ 64

28386 Citrate-MOPS Buffer 10packs ProteinimmobilizationtoUltraLinkBiosupport(Product#53110)

100mLof0.6Msodiumcitrate,0.1MMOPS,pH7.5

$ 58

28390 MES Buffered Saline 10packs Crosslinkingusingcarbodiimide(EDC,Product#22980)

500mLof0.1MMES,0.9%NaCl,pH4.7 $112

28372 Phosphate Buffered Saline (PBS) 40packs Crosslinkingandbiotinylationrequiringamine-freebuffer

500mLof0.1Msodiumphosphate,0.15MNaCl,pH7.2

$135

28374 Modified Dulbecco’s PBS 40packs WashbuffersandantibodydiluentsforELISA,Westernblottingandotherimmunoassays

500mLof8mMsodiumphosphate,2mMpotassiumphosphate,0.14MNaCl,10mMpotassiumchloride,pH7.4

$ 96

2837928376

Tris Buffered Saline 10packs40packs

WashbuffersandantibodydiluentsforELISA,Westernblottingandotherimmunoassays

500mLof25mMTris,0.15MNaCl,pH7.2

$ 61$134

28378 Tris-Glycine-SDS Buffer 40packs RunningbufferforTris-Glycinegelelectrophoresis

500mLof25mMTris,192mMglycine,0.1%SDS,pH8.3

$120

28398 Tris-HEPES-SDS Running Buffer 10 10packs RunningbufferforTris-HEPESelectrophoresiswithPrecise™PrecastGels

500mLof100mMTris,100mMHEPES,3mMSDS,pH8±0.25

$ 36

28380 Tris-Glycine Transfer Buffer 40packs Runningbufferforgeltomembraneelectrophoretictransfer

500mLof25mMTris,192mMglycine,pH8(use20%methanoltodissolve)

$120

Need a larger quantity?

Call our Bulk and Custom department at 1-800-874-3723

or +1 815-968-0747 ext. 300 for pricing and information. Or, visit

www.thermoscientific.com/protein-custom


Thermo Scientific Concentrated Liquid BuffersSave counter space and time.

Pierce10Xand20XConcentratedBuffersarereadytousewithouthavingtoreconstitutewithultrapurewaterand,inthecaseofTris-GlycineBuffer,methanol.Buffersaredesignedforuseindialysis,cross-linking,enzymeassays,ELISAs,immunohistochem-istry,proteinplate-coating,biotinylationandotherapplications.Keepourconcentratedbufferscloseathandtocoveryourlab’sresearchneeds.

Highlights:•Easy to use–nopacketstoopenandnopowdertodissolve• Increased accuracy–eliminatesthepossibilityofpowder

remaininginapacket• Saves time–10Xor20Xconcentrationeliminatestimespent

waitingforpowdertodissolve• Saves space–storageasconcentratedstockminimizesbench

spaceneededforlargevolumesolutions

PBS Tris-Glycine-SDS Tris-Hepes-SDS


Description

Pkg. Size

Applications

1X Formulation

U.S. Price

28341 20X Borate Buffer 500mL IdealforproteinmodificationproceduresrequiringaminefreebufferatalkalinePH

500mLof50mMborate,pH8.5 $68

28344 20X Modified Dulbecco'sPBS Buffer

500mL UsedforwashbuffersandantibodydiluentsinapplicationssuchasELISA,Westernblottingandotherimmunoassays

8mMSodiumPhosphate,2mMPotassiumPhosphate,0.14MNaCl,100mMKCl,pH7.4

$68

28346 20X Modified Dulbecco'sPBS Tween-20 Buffer

500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays

8mMSodiumPhosphate,2mMPotassiumPhosphate,0.14MNaCl,100mMKCl,0.05%Tween-20,pH7.4

$68

28348 20X Phosphate Buffered Saline 500mL Itsionicstrengthmakesitidealforcrosslinkingandbiotinylationrequiringaminefreebuffer

0.01MSodiumPhosphate,0.15MNaCl,pH7.5

$68

28352 20X PBS Tween-20 Buffer 500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays

0.01MSodiumPhosphate,0.15MNaCl,0.05%Tween-20,pH7.5

$68

28354 20X TAE Buffer 500mL Historicallythemostcommonbufferusedforagarosegelelectrophoresisintheanalysesofnucleicacids

0.04MTris,0.04MAcetate,0.001MEDTA,pH8.2-8.4

$68

28355 10X TBE Buffer 1L Oftenusedforagarosegelelectrophoresisintheanalysisofnucleicacids

0.089MTris,0.089MBorate,0.002MEDTA,pH8.2-8.4

$65

28358 20X TBS Buffer 500mL UsedforwashbuffersandantibodydiluentsinapplicationssuchasELISA,Westernblottingandotherimmunoassays

25mMTris,0.15MNaCl,pH7.2 $68

28360 20X TBS Tween-20 Buffer 500mL AwashbufferforELISA,WesternandotherImmunoassaysaswellasablockingbufferforplatebasedassays

25mMTris,0.15MNaCl,0.05%Tween-20,pH7.5

$68

28362 20X Tris/HEPES/SDS Buffer 1L ArunningbufferforTris-HEPESelectrophoresiswithPrecisePrecastGels

0.025MTris,0.192MGlycine,0.1%SDS,pH8.5

$45

28363 10X Tris-Glycine Buffer 1L Greatforelectrophoretictransferfromgeltomembrane

0.025MTris,0.192MGlycine,pH8.5 $45

28368 20X Tris/HEPES/SDS Buffer 500mL ArunningbufferforTris-HEPESelectrophoresiswithPrecisePrecastGels

0.1MTris,0.1MHEPES,3mMSDS,pH8+0.25

$68


Spin Cups and Columns

ThermoScientificPierceSpinColumnsareconvenienttoolsformanipulatingsmallvolumesofaffinitysupports(5–500μL)forproteinpurification.Addtheaffinityresinandsampletooneofthecolumns,useamicrocentrifugetoefficientlywashawaycontaminantsandeluteyourpurifiedsamplewithoutlosinganyresinintheprocess.Spincolumnsallowyoutoaffinity-purifymoreproteininlesstime!

Highlights:•Efficientwashingofsamplesmeansfewerwashesareneeded

toremovecontaminatingproteins•Efficientelutionofsamplesmeansmoreantigenand

co-precipitatedproteinsarerecovered•NoresinlossmeansmoreconsistentIPandco-IPresults•NoneedtodecantsupernatantfromIPorco-IPpellet•SpinprotocolsdrasticallyreducethetimerequiredforIPsandco-IPs•Lowprotein-bindingpolypropylenecolumnconstruction

minimizesnonspecificbinding

Spin Cups – Paper FilterPaper filter with collection tubes.

Highlights:•Paperfiltersareresistanttoclogging

fromcellulardebris•ColumnVolume:600μL•ResinVolume:20–300μL•FilterType:Paper,~10μmporesize•CapType:Collectiontubecapfitsontoinsertedspincup

Spin Cups – Cellulose Acetate FilterCellulose acetate filter with collection tubes.

Highlights:•UsedinourIPandCo-IPKits•ColumnVolume:800μL•ResinVolume:20–400μL•FilterType:Celluloseacetate,0.45μmporesize•CapType:Collectiontubecapfitsontoinsertedspincup

Spin Columns – Screw CapScrew cap with Luer-Lok® Adaptors.

Highlights:•Luer-LokAdaptorsallowthesecolumns

tobeusedforsyringe-basedpurifications•ColumnVolume:900μL•ResinVolume:20–400μL•FilterType:Polyethylene,~10μmporesize•Small&largefritoptionsfordifferentsamplesizes•CapType:O-ringscrewtopcaps;press-inbottomplugs

Spin Columns – Snap CapSnap cap with collection tubes.

Highlights:•UsedintheCellSurfaceProteinIsolation

andGlycoproteinIsolationKits•ColumnVolume:1,000μL•ResinVolume:20–500μL•FilterType:Polyethylene,~30μmporesize•CapType:Snapcaponcolumn(nocaponcollectiontube);

press-onbottomcaps

Micro-Spin ColumnsHighlights:•ColumnVolume:400μL•ResinVolume:5–100μL•FilterType:Polyethylene,~30μmporesize•CapType:O-ringscrewtopcaps;press-on

bottomcaps


Description

Pkg. Size

U.S. Price

69700 Spin Cups – Paper Filter Includescupsandcollectiontubes

50/pkg $109

69715 Microcentrifuge Tubes CollectionTubesforProduct#69700

72/pkg $ 32

69702 Spin Cups – Cellulose Acetate Filter Includescupsandcollectiontubes

50/pkg $108

69720 Microcentrifuge Tubes CollectionTubesforProduct#69702

72/pkg $ 32

69705 Spin Columns – Screw Cap with Luer-Lok Adaptors Includes:SpinColumns,ScrewCapsandColumnPlugs Luer-LokAdaptors LargeFrits(6.8mmdiameter10μmporesize) SmallFrits(2.7mmdiameter10μmporesize) LargeandSmallFritTools

Kit25each5each25each25each1each

$ 99

69725 Spin Columns – Snap Cap with Collection Tubes Includes:SpinColumnsandBottomCaps CollectionTubes

Kit50each100each

$109

89879 Micro-Spin Columns 50/pkg $ 55



Disposable Plastic Columns

Automatic “stop-flow” action provided by porous polyethylene discs prevents column beds from drying out.

Highlights:•Suppliedcompletewithporouspolyethylenediscs,stoppers,endcaps•Compatiblewithmosttypesofaqueousbuffereluentscommonly

usedinchromatography•Canbepre-packedandstoreduntilneeded


Description

Pkg. Size

U.S. Price

29920 Disposable Polystyrene ColumnsIdealforpacking0.5–2.0mLbedvolumes.

100/pkg $144

29922 Disposable Polypropylene Columns Idealforpacking1–5mLbedvolumes.

100/pkg $174

29924 Disposable Polypropylene ColumnsIdealforpacking2–10mLbedvolumes.

100/pkg $190

29923 Disposable Polypropylene Funnels BufferreservoirsthatfitProduct#’s29920,29922and29924.

50/pkg $109

29925 Disposable Column Trial Pack IncludesaccessoriesplustwoeachofProduct#’s29920,29922and29924andoneofProduct#29923.

TrialPack $ 63

Centrifuge Columns

Efficientlyhandleawidevarietyofresinvolumesforaffinitypurification!ThermoScientificPierceCentrifugeColumnsareconvenienttoolsforhandling40μL–10mLofanaffinitypurificationsupport.Addtheaffinityresintooneofthepolypropylenecolumns,removethetwist-offbottomandallowtheresintopackitself.Thenaddyoursampleandallowittobindtothesupport.Useacentrifugetoefficientlywashawayanycontaminantsandeluteyourpurifiedprotein.

PierceCentrifugeColumnsallowyoutouseaspin-columnformatinadditiontotraditionalgravityflowtoreducethetimerequiredforcolumnwashingandelution.Thisacceleratessampleprocessingtimeandmakesmultiple-sampleprocessingpossible.Centrifugecolumnsallowyoutoaffinity-purifymoreproteininlesstime!

Centrifugecolumnsaremadefromlowprotein-bindingpolypropyleneforcompatibilitywithproteinpurification,andtheyfitintostandardcentrifugetubesforuseinanycentrifuge.

Applications for Centrifuge Columns:•Affinitypurification/affinitychromatography•Immunodepletion•Spindesalting

0.8mL Centrifuge ColumnsHighlights:•TotalVolume:800μL•ResinVolume:40–400μL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandardmicrocentrifugetubes (e.g.,Product#69720)•CapType:O-ringscrew-topcap•Twist-offbottom

2mL Centrifuge ColumnsHighlights:•TotalVolume:5mL(2mLresinbed,3mLreservoir)•ResinVolume:2mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard15mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal

5mL Centrifuge ColumnsHighlights:•TotalVolume:8mL(5mLresinbed,3mLreservoir)•ResinVolume:5mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard15mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal

10mL Centrifuge Columns Highlights:•TotalVolume:22mL(10mLresinbed,12mLreservoir)•ResinVolume:10mL•FilterType:Polyethylene,~30μmporesize•ReceiverTube:Fitsstandard50mLconical centrifugetubes•CapType:Screw-topcap•Twist-offbottomandpress-oncaptoreseal


Description

Pkg. Size

U.S. Price

89868 Centrifuge Columns, 0.8mL capacity Includes:PierceCentrifugeColumns ScrewCaps

Kit50each50each

$ 62

89869 Centrifuge Columns, 0.8mL capacity Includes:PierceCentrifugeColumns ScrewCaps

Kit4x50each4x50each

$192

89896 Centrifuge Columns, 2mL capacity Includes:PierceCentrifugeColumns ScrewCapsandTips

Kit25each25each

$ 39


Kit25each25each

$ 44


Kit25each25each

$ 49

69707 Column Extender Fits89896,89897and89898.Increasescolumncapacityby35mL

10each $ 28

5ml

4ml

3ml

2ml

1ml

10ml 9ml

8ml 7ml

6ml 5ml

4ml 3ml

2ml 1ml


Fusion Protein Purification

CulturesofE. coliorPicchiaarecommonvehiclesforproteinexpression.Theyarelowcostandlowmaintenanceplatformswhichcanbeeasilyscaleduptodeliverproteinatmilligramtogramyields.Thesemicrooganismsarealsoveryeasytotrans-formwithaDNAvectorcontainingthegeneofinterest.Typically,researchersusecommonexpressionvectorswhichpossesstheproperpromoterelementsforexpressionandinclusionofanaffinitytagsequence.TheaffinitytagsequenceisclonedinframewiththeDNAsequenceofthetargetprotein,andwillflankeithertheN-orC-terminus.Thetwomostcommonaffinitytagsarepolyhistidine(6xHis)andglutathione-S-trasferase(GST).

Polyhistidine Purification

Cobalt-histidine binding.

Thepolyhistidinetagisasequenceoffivetoninehistidineaminoacidsattachedtotheterminusofatargetprotein.Thepolyhistidinetagispurifiedusingimmobilizedmetalaffinitychromatography(IMAC).Forhistidinetagpurification,eithernickelorcobaltisimmobilizedontoasolidchromatographyresin.Whilethetwometalscanbeusedinterchangeably,typicallynickelhasahigherbindingcapacitywhereascobaltbindslessnon-specificproteintodeliverapurerfinalprotein.

IMACresinsworkbychargeinteractionswiththenitrogenatomsonthehistidineaminoacidsidechaintobindandimmobilizethehistidine-taggedproteinfromacelllysate.Theincorporationofmultiplehistidineresiduesasanaffinitytagisdesignedtoimprovethischargeassociation.BecauseIMACaffinityforhistidineresi-duesisnotdependentonthesecondarystructureoftheprotein,IMACpurificationcanbeperformedunderdenaturingconditions.IMACpurificationis,however,sensitivetopHandthepresenceofchelatorsandreducingagents.SeeTable1foralistofcommoninterferingagentsandtheirconcentrationtoleranceforbothnickelandcobaltresin.



Nickel-histidine binding.

Table 1. Common interfering agents for Ni-NTA and cobalt resins.

Reagent Tolerance for Ni-NTA resin Tolerance for Cobalt resin

ß-Mercaptoethanol 10mM 10mM

CHAPS 1% 1%

Ethanol 30% 30%

Ethyleneglycol 30% 30%

HEPES 50mM 50mM

Glycerol 20% 20%

Guanidinium 6M 6M

Imididazole 500mM 200mMatpH7.0-8.0forelution

KCl 500mM 500mM

MES 20mM 20mM

MOPS 50mM 50mM

NaCl 1.0M 1.0M

NP-40 1% 1%

SDS 1% 1%

TRIS 50mM 50mM

Triton®-X-100 <1% <1%

Urea 8M 8M

Onceimmobilized,imidazoleisusedtodisruptthechargeattractionsbetweentheimmobilizedmetalaffinitychromatographyresinandthehistidine-taggedprotein.Theelutedhistidine-taggedproteincanbeeasilycleaned-upusingadesaltingcolumnordialysiscassettetoremoveimidazole.Oftentraceamountsofimidazoleareincludedintheproteinbindingandwashstepstocompeteawaybindingofendogenousproteinswithmultiplehistidines.

Glutathione-S-transferase Purification

GST binding.

GSTisa26kDaendogenousenzymefoundinbothprokaryotesandeukaryotes.InthecellGSTdetoxifiesreactiveoxygenspeciessuchasfreeradicalsandperoxides.GSTperformsthistaskbyneutralizingreactiveoxygenspecieswiththeantioxidantglutathione(GSH).GSHisanendogenoustripeptide(Glu-Cys-Gly)containingacysteineresidue,whosesulfhydrylsidechainfunctionsasareducingagent.

GSTmakesanidealaffinitytagbecauseofitsstrongbindingtoreducedglutathione.GSTpossessnumeroustyrosineresiduesintheintheGSHbindingpocket,andoneofthesetyrosineshydrogenbondstothesubstrateglutathioneformingastablecomplex.In vivo,GSTwouldtransferglutathionetoareactiveoxygenspeciesandneutralizeit.

Asaresearchtool,scientistshavetakenadvantageofthestronginteractionbetweenGSTandGSHtoselectivelyextractrecom-binantproteins.Inthisstrategy,glutathioneisimmobilizedontoasolidsupport(typicallyanagarosebead)andtheaminoacidsequencefortheenzymeGSTisclonedintoeithertheC-orN-terminusofthetargetgene(commonlyusingthepGEX®-seriesofexpressionvectors).AfterbindingoftheGST-taggedfusionproteintotheimmobilizedglutathioneagarose,excessreducedglutathioneisintroduced(typicallybetween10-50mM)tocompeteoffthetargetprotein.Again,simpledialysisordesaltingcolumnscanbeusedtocleanupthefinalGST-taggedprotein.Forreasonsthathavenotbeenfullycharacterizedintheliterature,thestruc-tureoftheGSTfusiontagoftendegradesupondenaturationandreductionforproteingelelectrophoresis(e.g.,SDS-PAGE).Asaresult,electrophoresedsamplesoftenappearasaladderoflowerMWbandsbelowthefull-sizedfusionprotein.

Incontrasttopolyhistidinepurification,GSTpurificationrequiresthetargetproteinmaintainnativetertiarystructure.Additionally,theGSTtagisquitelarge(26kDa)comparedtothesixhistidineswhichcompriseatypicalpolyhistidinetag.Tocircumventtheproblemsassociatedwitha26kDaappendage,selectiveproteasesareusedtocleavetheGSTtagfromtheGST-fusionprotein.TheseproteasesincludeFactorXa(Product#32520)orthrombin.


His-Tagged Protein Purification Resin

A cost-effective Ni-NTA resin is now available.

Theexpressionandpurificationofrecombinantproteinsiscentraltoproteinregulation,structureandfunctionstud-ies.Themajorityofrecombinantproteinsareexpressedasfusionswithshortaffinitytagswiththemostpopularbeingthepolyhistidine(6xHis)tag.ThemethodusedtopurifyrecombinantHis-taggedproteinsisimmobilizedmetalaffinitychromatography(IMAC),consistingofchelatingresinschargedwitheithernickelorcobaltionsthatcoordinatewiththehistidinesidechains.ThenewThermoScientificHisPurNi-NTAResin

complementstheHisPur™CobaltResin.Ni-NTAresinsarethemostcommonIMACresinchoicefor6xHis-taggedproteinpurificationsbecauseofthefourmetal-bindingsitesonthechelate,whichenableshigh-proteinbindingandlow-metalionleaching.

Highlights:• High capacity–bindupto60mgof6xHis-taggedproteinper

milliliterofresin• Versatile–purifyproteinsusingnativeordenaturingconditions• Compatible–usewithThermoScientificCellLysisReagents

andavarietyofbufferadditives• Flexible–availableinmultipleformats,includingbulkresin,spin

columnsandchromatographycartridges•Cost-effective–reusethesamebatchofresinatleastfivetimes•Easy to use–pre-formulatedbuffersavailableforkitformats

His-taggedgreenfluorescentprotein(GFP)wasrecoveredinsimilarorgreaterpurityandyieldcomparedtoothercommerciallyavailablenickel-chelatedresins(Figure1).HisPurNi-NTAResinisalsocost-effectivebecauseitcanbeusedatleastfivetimeswithoutlossinperformance(Figure2).Theresinisamenabletochromatography-cartridgeformat,resultinginasharpelutionpeakevidentinthechromatogram(Figure3).WhenHisPurNi-NTAResiniscomparedtoHisPurCobaltResin(Product#90090)andNi-IDAresinusingthebatch-bindmethod,theresultsdependonproteinexpressionlevel(Table2andFigure4).Whenpurifyingalow-expressionprotein,suchas6xHis-AIF2,thereisadramaticdifferenceinpurity.HisPurCobaltResinyieldsthepurestproteinfollowedbyHisPurNi-NTAResinwithNi-IDAbeingtheleastpure.Whenpurifying6xHis-GFP,ahighexpresser,therewasminimaldifferencebetweenHisPurNi-NTAandCobaltResins.Similarresultswereobtainedusingspinpurification(datanotshown).

M

10

15

2520

37

50

75

100

150

250250kDa

L HisPur Ni-NTA

Supplier Q

Supplier C

Ni-IDA

6xHis-GFP

Figure 1. Thermo Scientific HisPur Ni-NTA resin is comparable to or performs better than other suppliers’ nickel resins.Bacteriallysate(12mgtotalprotein)containingover-expressed6xHis-greenfluorescentprotein(GFP)wasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.ThesameamountoftotalproteinwasappliedtoSupplierQ,SupplierCandNi-IDAresinsperthemanu-facturers’instructions.Gellaneswerenormalizedtoequivalentvolume.M =molecular-weightmarkerandL =lysateload.



M

First Use

L FT W E

Fifth Use

L FT W E

10

15

25

20

37

50

75

100

150kDa

6xHis-Protein L

Figure 2. Thermo Scientific HisPur Ni-NTA Resin can be used at least five times without losing performance. Bacteriallysate(20mgtotalprotein)con-tainingover-expressed6xHis-ProteinLwasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.Beforeeachreuse,theresinwaswashedwith20mmMESbuffer,0.1MNaCl;pH5(1mL)andfollowedbyawaterwash(1mL).Gelslaneswerenormalizedtoequivalentvolume.M =molecular-weightmarker,L =lysateload,FT =flow-throughandE =elution.

280 nm485 nm

0

500

1000

1500

2000

2500

3000

3500

mAU

Flow-through

Wash

0 20 40 60 80 100 120mL

6xHis-GFP

Figure 3. Thermo Scientific HisPur Ni-NTA Chromatography Cartridges provide an automatable solution for obtaining high-purity proteins. Bacteriallysate(140mgtotalprotein)containingover-expressed6xHis-GFPwasdilutedwithequilibrationbufferandappliedtoaHisPurNi-NTAChromatographyCartridgeataflowrateof1mL/min.ThecartridgewaswashedwithPBS,60mmimidazoleuntilthebaselineabsorbancewasreached.6xHis-GFPwaselutedwithPBS,300mmimidazole.Elutionwasmonitoredat280nm(blueline)and485nm(redline).

Table 2. Elution fractions were analyzed for protein content using the Thermo Scientific Coomassie Plus (Bradford) Protein Assay Kit (Product # 23236). Purity was determined by analyzing the stained SDS-PAGE gel (Figure 4) with densitometry software.

Sample Resin Total yield (mg) Purity

6xHis-AIF2 HisPurNi-NTA 0.5 32%

Ni-IDA 0.5 25%

HisPurCobalt 0.4 49%

6xHis-GFP HisPurNi-NTA 0.8 90%

Ni-IDA 0.6 52%

HisPurCobalt 0.7 91%

M

10

15

2520

37

50

75

100

150

250kDa

L HisPur N

i-NTA

Ni-IDA

HisPur C

obalt

L HisPur N

i-NTA

Ni-IDA

HisPur C

obalt

6xHis-GFP

6xHis-AIF2

Figure 4. Thermo Scientific HisPur Cobalt Resin produces the most pure results. Bacteriallysatecontainingover-expressed6xHis-AIF2(6mgtotalprotein)or6xHis-GFP(4mgtotalprotein)wasappliedtoHisPurNi-NTAResin(200μL)andpurifiedbythebatch-bindmethodasdescribed.ThesameamountoftotalproteinwasappliedtoNi-IDAandHisPurCobaltResinsandpurifiedasdescribedintheinstructions.Gelslaneswerenormalizedtoequivalentvolume.M =molecular-weightmarker,L =lysateload.

TheHisPurNi-NTAResinoffersacost-effectivealternativetoothercommerciallyavailablenickel-IMACresins,withoutcompro-misingyield,purityorperformance.TheHisPurNi-NTAandCobaltResinsareavailableinmultipleformatstoaccommodateavarietyofapplicationsandpurificationvolumes.

Ordering Information

Product # Description Pkg. SizeU.S. Price

88221 HisPur Ni-NTA Resin 10mL $ 85

88222 HisPur Ni-NTA Resin 100mL $ 579

88223 HisPur Ni-NTA Resin 500mL $2275

88224 HisPur Ni-NTA Spin Columns, 0.2mL 25columns $ 155

88225 HisPur Ni-NTA Spin Columns, 1mL 5columns $ 99

88226 HisPur Ni-NTA Spin Columns, 3mL 5columns $ 150

88227 HisPur Ni-NTA Purification Kit§, 0.2mL 25columns $ 269

88228 HisPur Ni-NTA Purification Kit§, 1mL 5columns $ 253

88229 HisPur Ni-NTA Purification Kit§, 3mL 5columns $ 269

90098 HisPur Ni-NTA Chromatography Cartridges, 1mL

5cartridges $ 155

90099 HisPur Ni-NTA Chromatography Cartridges, 5mL

2cartridges $ 200

§ For complete ordering information and kit components, please visit www.thermoscientific.com/pierce


Cobalt Resin, Spin Columns and Chromatography Cartridges

Specific, fast and gentle purification of His-tagged proteins.

ThepreferredmethodforpurifyingrecombinantHis-taggedproteinsisimmobilizedmetalaffinitychromatography(IMAC).Traditionally,chelatingchromatographyresinsarechargedwitheithernickelorcobaltionsthatcoordinatewiththehistidinesidechainsinthe6xHis-tag.HisPurCobaltResinisatetradentatechelatingresinchargedwithcobaltthatbindsHis-taggedproteinswithhighspecificityandreleasesthemunderlowerimidazoleconcentrationsthanrequiredwithnickelresins(seeFigure5).HisPurCobaltResincanbeusedtoobtainhigh-purityproteinswithnometalcontamination.

TheHisPurChromatographyCartridgesareconvenient,reliableandready-to-usepre-packeddevicesfortheisolationofproteinsinsolutionandpurificationofHis-taggedfusionproteins.TheHisPurCartridgesarecompatiblewithautomatedLCinstrumen-tation,suchastheÄKTA®andBioLogicSystems,andadapttomanualsyringeprocessing.

Wash Elution

16.5

2532

47

110

210kDa M F 1 2 3 1 2 3 4 5 L

6xHis-GFP

Thermo Scientific HisPur Cobalt Resin is specific for His-tagged proteins and allows mild, efficient elutions.Bacteriallysate(1.0mgtotalprotein)wasappliedtoa200μLbedvolumeofHisPurCobaltResininaspincolumn.Gellaneswerenormalizedtoequivalentvolume.M =MolecularWeightMarkers(Product#26691),L =lysateloadandF =flow-through.

Highlights:•High purity–obtainpureproteinwithoutoptimizingimidazole washingconditions•Specificity–cobalt:chelatebindingcorebindsfewercontaminants, resultinginlowerbackgroundthannickel(seeTable3)•Low metal leaching–nometalcontaminationinelutedsample•Versatility–purifyproteinsundernativeordenaturingconditions; compatiblewithcelllysisreagentsandavarietyofbufferadditives•Flexibility–availableasbulkresinorpredispensedcolumns compatiblewithbothspinandgravity-flowformats•Cost effective–reuseordiscard•Superior–performsbetterthanothercommerciallyavailable IMACResins(seeFigure5)

Co2+

1

GFP β-gal Protein L

2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 L

Ni2+ Ni2+Co2+ Ni2+Co2+

Figure 5. Thermo Scientific HisPur Cobalt Resin outperforms other IMAC resins. ComparableyieldandhigherpurityobtainedwithHisPurCobaltResin(lane1)comparedtootherIMACresins(lanes2to6).Celllysatescontainingover-expressedrecombinant6xHis-taggedproteinwerepreparedinB-PERBacterialProteinExtractionReagent(Product#78243)andProteaseInhibitors(Product#78410).ProteinconcentrationsweredeterminedbyCoomassiePlusProteinAssay(Product#23238).E. coli lysatescontainingover-expressedHis-taggedGFP, β-galactosidaseorProteinLwereappliedto0.2mLbedvolumesofeachIMACresininspincolumnformat.Binding,washandelutionbufferswerepreparedandusedpereachmanufacturers’instructions.ThefirstelutionfractionforeachIMACresinwasanalyzedbySDS-PAGEandproteinpuritydeterminedbydensitometry.Gellaneswerenormalizedtoequivalentvolume.Lanes: 1=HisPurCobaltResin,2=supplierCcobaltresin,3=supplierScobaltresin,4=supplierGnickelresin,5=supplierQnickelresin,6=Ni-IDAandL=lysateload.



Table 3. Thermo Scientific HisPur Cobalt Resin yields more His-tagged protein and higher purity than other Co2+ and Ni2+ IMAC Resins.

His-GFP His-β-Gal His-Protein L

Yield(μg)* Purity(%)** Yield(μg)* Purity(%)** Yield(μg)* Purity(%)**

ThermoScientificHisPurCobaltResin 298 87 78 93 42 77

SupplierCCobaltResin 206 78 26 90 35 76

SupplierSCobaltResin 211 85 27 65 38 77

SupplierGNickelResin 239 84 42 83 29 68

SupplierQNickelResin 242 85 24 48 30 72

Ni2+-IDAResin 70 37 6 16 17 46

* Recovered from a 5mg total protein load (total protein yield x purity). ** Purity of the first elution fraction.

HisPur Cobalt Cartridge Performance Data:

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20 25Chromatography Progress (mL)

Flow-through Wash Elution

Abs

orba

nce

280n

m (m

AU

)

Flow-through (FT)M S Wash Elution

Electrophoresed Fractions

Chromatography Profile

Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Cobalt Cartridge. His-taggedgreenfluorescentprotein(GFP)wasextractedfromE. coliusingThermoScientificB-PERBacterialProteinExtractionReagentinPhosphateBuffer(Product#78266)containingHaltProteaseInhibitorCocktail,EDTA-Free(Product#78415).Thelysatewasdiluted1:1withequilibration/washbuffer(50mMsodiumphosphate,300mMsodiumchloride,10mMimidazole,pH7.4)andappliedtoaThermoScientificHisPurCobaltChromatographyCartridgeataflowrateof0.3mL/min.Thecartridgewaswashedwithequilibration/washbufferuntilthebaselineabsorbanceatA280wasreached.His-taggedGFPwaseluted(50mMsodiumphosphate,300mMsodiumchloride,150mMimidazole;pH7.4)andselectedfractionswereanalyzedbySDS-PAGEandThermoScientificGelCodeBlueStainReagent(Product#24592).M =MWMarker;S=non-fractionatedlysate;FT =flow-through.


Description

Pkg. Size

U.S. Price

89967 HisPur Cobalt Spin Columns, 0.2mL 25columns $ 160

89968 HisPur Cobalt Spin Columns, 1mL 5columns $ 105

89969 HisPur Cobalt Spin Columns, 3mL 5columns $ 155

89964 HisPur Cobalt Resin 10mLbottle $ 88

89965 HisPur Cobalt Resin 100mLbottle $ 600

89966 HisPur Cobalt Resin 500mLbottle $2347

90090 HisPur Purification Kit, 0.2mL 25columns $ 277

90091 HisPur Purification Kit, 1mL 5columns $ 262

90092 HisPur Purification Kit, 3mL 5columns $ 277

90093 HisPur Chromatography Cartridges 5x1mL $ 160

90094 HisPur Chromatography Cartridges 2x5mL $ 267



High-quality purification of GST-fusion proteins

Multiple formats available to meet your purification needs

PurificationofglutathioneS-transferase(GST)fusionproteinsusingglutathioneagarosebeadsprovidesone-step,high-purityaffinitypurification.TheThermoScientificPierceGlutathioneAgaroseiscomposedof6%crosslinkedbeadedagarosewithglutathione(GSH)immobilizedbyitscentralsulfhydryl.GST-fusionproteinsarepurifiedwithhighyieldbecauseofthe12-atomGSHlinkerthatminimizessterichindrance.ThePierceGlutathioneAgaroseisavailableinresinslurrypackages,spincolumns,completepurificationkitsandFPLC-readychromatographycartridges.

Highlights:• High capacity–bindsatleast40mgofpureGSTpermilliliter

ofresin• Cost-effective–resiniseconomicallypricedandcanbereused

atleastfivetimeswithoutreductioninperformance• High yield and purity–eachmilliliterofresincanpurify≥ 10mg

ofGST-taggedproteinfromlysateswith>90%purity• Compatible–validatedandeffectivewithThermoScientific

CellLysisReagents• Versatile–purifylysatesorpulldownprotein-proteininteractions• Flexible–availableinmultipleformats

GlutathioneS-transferaseandpolyhistidinearethemostcommonlyusedproteintagsforaffinitypurification.GSTproducescleanerpurificationsthanhistidinetagsandstabilizesoverexpressedfusionproteins.GSTbindsspecificallytoreducedGSHinnear-neutral,nondenaturingconditions.TheboundGST-taggedproteiniseasilydissociated(eluted)fromGSHresinbycompetitivedisplacementwithabuffercontainingfree,reducedGSH.Alternatively,ifthefusionproteinisdesignedwithaproteasecleavagesite,itmaybereleased(cleaved)fromthecolumnattheGSTtagjunctionusingthrombin,HRV3CproteaseorFactorXa.

ThePierceGlutathioneAgaroseishighlyeffectiveforpurifyingavarietyofGST-fusionproteins(Figure6).ThepurityandyieldofGST-fusionproteinrecoveryiscomparabletoorbetterthanglutathioneresinsfromothersuppliers(Figure7).Theresiniscomposedof6%crosslinkedbeadedagarose,makingitastructurallystableandresilientaffinitysupportthatenablesrepeateduses.ThePierceGlutathioneAgaroseisalsoamenabletochromatographycartridgeformat,producingbaselineresolutionandasharpelutionpeak(Figure8)withnovisibleresinbedcompression.

GST-Pak1

kDa M L FT W1 W2 W3 E1 E2 E3

210

11080

47

3225

16.5

GST-ERK

M L FT W1 W2 W3 E1 E2 E3kDa

210

11080

47

3225

16.5

GST-Bid

M L FT W1 W2 W3 E1 E2 E3kDa

210

11080

47

3225

16.5

Figure 6. The Thermo Scientific Pierce Glutathione Agarose effectively purifies different GST-fusion proteins.ThreeGST-fusionproteinswereexpressedinE. coliandextractedwithThermoScientificB-PERReagentwithEnzymes(Product#90078).GST-Pak1andGST-Bidwerepurifiedusingthespinformat,andGST-ERKwaspurifiedbygravityflow.GST-Pak1andGST-Bid,2.4mgand11mgoftotalprotein,respectively,werepurifiedwitha0.2mLPierceGlutathioneSpinColumn(Product#16106)perkitinstructions.ForGST-ERK,11.2mgoftotallysateproteininbuffer(50mMTris,150mMNaCl;pH8.0)wasappliedtoa0.5mLbedofPierceGlutathioneAgarose(Product#16100)inadripcolumnandelutedwith125mMTris,150mMNaCl,10mMglutathioneatpH8.0.Chromatographyfractionsweresep-aratedbySDS-PAGEandstainedwithThermoScientificGelCodeBlueStainReagent(Product#24590).M =Molecularweightmarkers;L =Lysateload; FT =Flow-through;W =Wash;E =Elution.


kDa

250150100

755027

25201510

M L FT

ThermoScientific

GEHealthcare Qiagen Clontech Sigma

E FT E FT E FT E FT E

VendorThermo

ScientificGE

Healthcare Qiagen Clontech Sigma

Yield 537μg 562μg 285μg 299μg 410μg

Purity 93% 93% 90% 91% 94%

Figure 7. Thermo Scientific Pierce Glutathione Agarose delivers high yield and high-purity GST-fusion proteins.E. coli lysate(14.4mgtotalprotein)con-taininganoverexpressedGST-fusionproteinwasincubatedwith50μLGSHresinfromvarioussuppliersandpurifiedpermanufacturers’instructions.TheamountofGSTelutedfromtheresin(yield)wasquantifiedbyThermoScientificCoomassiePlusProteinAssay.Puritywasassessedbydensitometryofthestainedgellanes.M=Molecularweightmarkers;L=Lysateload;FT=Flow-through;E=Elution.

0

0.0 10.0 20.0 30.0 40.0 50.0 ml

mAU

4000

Flow-through

Wash Elution

3000

2000

1000

Figure 8. The glutathione chromatography cartridges provide an automatable solution for GST-fusion protein purification.ClarifiedE. colilysate(50mg)containingoverexpressedGST-Pak1(34kDa)wasappliedtoa1mLglutathionechromatographycartridgeinbuffer(50mMTris,150mMsodiumchloride;pH8.0)ataflowrateof0.5mL/minuteusinganÄKTApurifier®System.Thecartridgewaswashedwiththesamebufferat1mL/minute.GST-Pak1waseluted(50mMTris,150mMsodiumchloride,10mMglutathione;pH8.0)at1mL/minute.

Ordering InformationSee catalog or www.thermoscientific.com/pierce for a complete description of products and kits.


16100 Pierce Glutathione Agarose 10mL $ 180

16101 Pierce Glutathione Agarose 100mL $ 975

16102 Pierce Glutathione Agarose 500mL $3000

16103 Pierce Glutathione Spin Columns, 0.2mL 25columns $ 175

16104 Pierce Glutathione Spin Columns, 1mL 5columns $ 175

16105 Pierce Glutathione Spin Columns, 3mL 5columns $ 315

16106 Pierce GST Spin Purification Kit, 0.2mL 25-columnkit $ 275

16107 Pierce GST Spin Purification Kit, 1mL 5-columnkit $ 275

16108 Pierce GST Spin Purification Kit, 3mL 5-columnkit $ 375

16109 Pierce Glutathione Chromatography Cartridges, 1mL

5cartridges $ 230

16110 Pierce Glutathione Chromatography Cartridges, 5mL

2cartridges $ 400


GST- and PolyHis-Tagged Pull-Down Assay Kits

Prepare to discover a new protein:protein interaction with your GST- or polyHis-tagged bait protein.

Identifyingandcharacterizingtheinteractionsofagivenproteinhasemergedasthemostvaluableinformationthatcanbedevelopedinthepost-genomicera.ThermoScientificPiercePull-DownKitscontainthenecessarycomponentstocaptureinteractingproteinsusingthepopularpull-downmethod.Theonlyitemyouprovideisanappropriatelytaggedfusionproteinasthe“bait.”OurPull-DownKitsaredesignedtoteachthemethodtothefirst-timeuserandtoshortenthetimetoaresultforthoseexperiencedinthismethod.

Step 3. Bind “prey” protein to immobilized “bait” protein.

Step 4. Wash away unbound protein. Step 5. Elute protein:protein interaction complex.

Step 2. Wash away unbound protein.Step 1. Immobilize the fusion-tagged “bait” from the lysate.

Step 6. Analyze protein:protein interaction complex on SDS-PAGE.

= Fusion Tag (GST or polyHis)

Bait

Prey

Marker PurifiedProtein:Protein

Interaction

AgaroseResin

Control

PurifiedBait

Displaced Interacting Complex

P re y P ro te inBait Protein Prey Protein

Bait Protein

Prey Protein-Containing Lysate

Bait Protein

Bait Protein

Bait Protein-Containing Lysate

Affinity Ligand(Glutathione or Co2+ Chelate)

AgaroseBead

Prey Protein

Bait Protein Prey Protein

Bait Protein Prey Protein

Glutathioneor

Imidazole

Spin

Spin

Highlights:• Providesacomplete,affordableandeasy-to-usestrategy

fordiscoveryofprotein:proteininteractions• Usescommonlaboratoryequipment(e.g.,microcentrifuges

andmini-gels)•Adaptstosingle-ormultiple-sampledemands•Suppliedcompletewithcelllysisbuffer• Flexibleprotocolaidsinthecaptureofweakortransient

interactions• Efficientrecoveryofinteractingcomplexes

Applications:•Discoveranewprotein:proteininteractionfromacelllysate• Confirmaputativeinteractionfromacelllysateorwitha

previouslypurifiedprotein• Extractprotein:proteininteractioninformationfrom in vitro

transcription/translationlysates

Generalized scheme for use of a Thermo Scientific Pierce Pull-Down Protein:Protein Interaction Kit using a GST-tagged or PolyHis-tagged protein as the “bait.”




Description

Pkg. Size

U.S. Price

21516 Pierce Pull-Down GST Protein Interaction Kit Sufficient materials for performing 25 pull-down assays using a GST-tagged protein as the bait.Includes:ImmobilizedGlutathione LysisBuffer Glutathione BupHTrisBufferedSaline SpinColumnsAccessoryPack CollectionTubesandCaps AccessoryPack

Kit

750μLsettledgel250mL1g1pack(500μL)27columns100tubes

$320

AcknowledgmentWegratefullyacknowledgeDr.YigongShiofPrincetonUniversityforsupplyingtheGST-BIR2-and9xHisSmac/DIABlO-expressingclones.Dr.Shi’slaboratoryisengagedinresearchaimedatunderstandingthestructuralandmolecularmechanismsinvolvedintumorigenesisandapoptosis.


Description

Pkg. Size

U.S. Price

21277 Pierce Pull-Down PolyHis Protein Interaction Kit Sufficient materials for performing 25 pull-down assays using a polyhistidine-tagged protein as the bait.Includes:ImmobilizedGlutathione LysisBuffer 4MImidazoleStockSolution BupHTrisBufferedSaline SpinColumnsAccessoryPack CollectionTubesandCaps AccessoryPack

Kit

750μLsettledgel250mL5mL1pack(500μL)27columns100tubes

$320

References1.Chai,J.,et al.(2000).Nature406,855-862.2.Kaelin,W.G.,et al.(1991).Cell64,521-532.3.Sambrook,J.andRussell,D.W.(2001).Molecular Cloning: A Laboratory Manual

3rd Edition.Chapter18:ProteinInteractionTechnologies,Protocol#3:DetectionofProtein-ProteinInteractionsusingtheGSTFusionProteinPull-DownTechnique.ColdSpringHarborLaboratoryPress.

4.Soutoglou,E.,et al. (2000).Mol. Cell 5, 745-751.

Lane # A. GST-Tag Pull-Down B. PolyHis-Tag Pull-Down

1 LysatefromE. coliexpressingGST-taggedBIR2(bait protein). LysatefromE. coliexpressing9xHis-taggedwild-typeSmac(bait protein).

2 Flow-throughfromthelysateinLane1boundtoanimmobilizedreducedglutathionesupportfor1hourat4°C.

Flow-throughfromthelysateinLane1boundtoanimmobilizedcobaltchelatesupportfor1hourat4°C.

3 Wash#1ofthesupport. Wash#1ofthesupport.

4 Wash#2ofthesupport.(Washes3-5notshown.) Wash#2ofthesupport.(Washes3-5notshown.)

5 LysatefromE. coli expressing9xHis-taggedwild-typeSmac(prey protein).

LysatefromE. coliexpressingGST-taggedBIR2(prey protein).

6 Flow-throughfromthelysateinLane5isallowedtointeractwiththeimmobilizedbaitfor1hourat4°C.

Flow-throughfromthelysateinLane5isallowedtointeractwiththeimmobilizedbaitfor1hourat4°C.

7 Wash#1ofthesupport. Wash#1ofthesupport.

8 Wash#2ofthesupport.(Washes3-5notshown.) Wash#2ofthesupport.(Washes3-5notshown.)

9 Baitcontrol.BaittreatedasdescribedinLanes1-8andsubsequentlyeluted.Nopreyadded–justbindingbuffer.

Baitcontrol.BaittreatedasdescribedinLanes1-8andsubsequentlyeluted.Nopreyadded–justbindingbuffer.

10 Preycontrol.PreytreatedasdescribedinLanes1-8andsubsequentlyeluted.Nobaitadded–justbindingbuffer.

Preycontrol.PreytreatedasdescribedinLanes1-8andsubsequentlyeluted.Nobaitadded–justbindingbuffer.

11 Elutionofbait:preycomplex(preparedinLanes1-8)fromthesupportwith100mmreducedglutathione.WesternblottingconfirmsthattheminorbandsobservedinLanes9and11aredegradationproductsofGST-taggedBIR2.

Elutionofbait:preycomplex(preparedinLanes1-8)fromthesupportwith250mmimidazole.

Validation of the Thermo Scientific Pierce Pull-Down Protein Interaction Kits using a known interacting pair.

1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 11A. B.


Covalent Immobilization of Ligands

Affinity chromatography uses the specific interactions between two molecules for the purification of a target molecule. In practice, a ligand having affinity for a target molecule is covalently attached to an insoluble support and functions as bait for capturing the target from complex solutions.

Theaffinityligandcanbevirtuallyanymoleculethatcanspecificallybindthetargetwithoutdisplayingsignificantnonspecificbindingtowardothermoleculesinthesolution.Ligandsthathavebeenusedforaffinityseparationsincludesmallorganiccompoundsthatareabletodockintobindingsitesonproteins,inorganicmetalsthatformcoordinationcomplexeswithcertainaminoacidsinproteins,hydrophobicmoleculesthatcanbindnonpolarpocketsinbiomole-cules,proteinswithspecificbindingregionsthatareabletointeractwithotherproteins,andantibodies,whichcanbedesignedtotargetanybiomoleculethroughtheirantigen-bindingsites.

Theconceptofusingimmobilizedaffinityligandstotargetbiomoleculeshasextendedbeyondchromatographicapplications.Affinityligandsarenowcoupledtolatexbeads,nanoparticles,macro-beads,membranes,microplates,arraysurfaces,dipsticksandahostofotherdevicesthatfacilitatethecaptureofspecificbiomolecules.Theapplicationofaffinitytargetingincludespurification,scavenging(orremovalofcontaminants),catalysis(ormodificationoftargetmolecules)andabroadrangeofanalyticalusestoquantifyatargetmoleculeinasamplesolution.

Designingcustomaffinitysupportsthatareabletotargetuniquebiomoleculesrequiresmethodstocovalentlylinkaligandtoaninsolublematrix.Regardlessoftheintendedapplication,thechemicalreactionsthatmakeligandattachmentpossiblearewellcharacterizedandfacilitatetheattachmentofbiomoleculesthroughtheircommonchemicalgroups.Thetypesoffunctionalitiesgenerallyusedforattachmentincludeeasilyreactivecomponentssuchasprimaryamines,sulfhydryls,aldehydes,carboxylicacids,hydroxyls,phenolicgroupsandhistidinylresidues.Usually,thesolid-phasematrixfirstisactivatedwithacompoundthatisreactivetowardoneormoreofthesefunctionalgroups.Theactivatedcomplexthencanformacovalentlinkagebetweentheligandandthesupport,resultinginligandimmobilization.

Thetypeoflinkagethatisformedbetweenthematrixandtheimmobilizedligandaffectstheperformanceoftheaffinitysupportinanumberofways.Alinkagethatallowsthecoupledligandtoleachfromthematrixwillresultincontaminationofthepurifiedproteinandshortentheusefullifeoftheaffinitysupport.Alinkagethatintroducesachargedfunctionalgroupintothesupportcancausenonspecificbindingbypromotingion-exchangeeffects.Alinkagethataltersthestructureofthematrixcanchangetheflowandbindingcharacteristicsofthesupport.Cyanogenbromide(CNBr)-activatedsupportsareinformativeasanexampleoftheseprinciples.Thispopularimmobilizationmethodresultsinalinkagethat:

1.Hasaconstantleakageofligandfromthematrixthatbecomesacontaminantinthepurifiedpreparation.

2.Includesachargedisoureagroupinthelinkage,resultinginnonspecificbinding.

3.Causesextensivecrosslinkingofthematrix,reducingtheabilityoflargemoleculestopenetrateintotheinterioroftheresin.

Weofferanumberofactivatedaffinitysupportsthataredesignedtocoupleligandsofeverytypeviastable,unchargedcovalentlinkagesthatavoidintroducingundesirablepropertiesintothesupports.Theactivationchemistryandprotocolshavebeenoptimizedtoensureexcellentcouplingyieldswithminimaleffortunderavarietyofconditions.Eachactivatedsupportcomeswithinstructionsforuseandliteraturereferencesasexamples.Theassociatedkitscontainallthecouplingbuffers,washbuffersandcolumnsnecessarytoperformtheligandimmobilizationandproduceasupportreadytoperformanaffinityseparation.

Covalent Coupling of Affinity Ligands to Chromatography Supports

22


Coupling Affinity Ligands through Amine Groups

Themostcommonfunctionaltargetforimmobilizingproteinmoleculesistheaminegroup,whichispresentonthevastmajorityofproteinsbecauseoftheabundanceoflysinesidechaine-aminesandN-terminala-amines.ThermoScientificAminoLinkCouplingResinandAminoLinkPlusCouplingResinarepreparedfromcrosslinkedagarosesupports,andtheyaredesignedtocreateastablelinkagebetweenaminegroupsandthesupportmaterial.AminoLink®Resinsareactivatedtocontainnumerousaldehydegroups,whichcanbeusedtoimmobilizeamine-containingligandsbyreductiveamination.

TheimmobilizationreactionusingreductiveaminationinvolvestheformationofaninitialSchiffbasebetweenthealdehydeandaminegroups,whichthenisreducedtoasecondaryaminebytheadditionofsodiumcyanoborohydride.Thecyanoborohydridereducingagentusedduringthecouplingprocessismildenoughnottocleavedisulfidesinmostproteins,anditwillnotreducethealdehydereactants–onlytheSchiffbaseintermediates.Itisbesttoavoidstrongerreducingagentssuchassodiumborohydridebecauseofthepotentialfordisulfidereductionoftheproteinandreductionofthealdehydesonthesupporttohydroxyls,effectivelyquenchingthereaction.Dependingonthetypeandamountofligandpresent,acouplingreactionusingreductiveaminationcanachieveimmobilizationyieldsofgreaterthan85%.

H

H2N–R

NaCNBH3

O N H

R

Aldehyde Group onThermo Scientific

AminoLink Coupling ResinAffinity Ligand Coupled

via Secondary Amine Bond

Anotheramine-reactivestrategythatcanbeusedforimmobilizationistheazlactoneringpresentinUltraLinkBiosupport.Aprimaryaminewillreactwithanazlactonegroupinaring-openingprocessthatproducesanamidebondattheendofafive-atomspacer.Thegroupisspontaneouslyreactivewithamines,requiringnoadditivesorcatalyststodrivethecouplingprocess.TheUltraLinkBiosupportissupplieddrytoensurestabilityoftheazlactonegroupsuntiluse.Addingaquantityofthesupporttoasamplecontainingaproteinorotheramine-containingmoleculecausesimmobilizationtooccurwithinaboutonehour.Forproteinimmobilizationathighyield,itisrecommendedthatthecouplingbuffercontainalyotropicsalt,whichfunctionstodrivetheproteinmoleculestowardthebeadsurface.Thisbringsthehydrophilicaminescloseenoughtotheazlactoneringstoreactquickly.ThesimplenatureofcouplingaffinityligandstotheUltraLinkBiosupportalongwithitsinherentlylownonspecificbindingmakesitoneofthebestchoicesforimmobilization.

O

N

O

CH3

CH3 NH

OHN

O

R

Azlactone Group onThermo Scientific

UltraLink BiosupportAffinity Ligand Coupled

via Amide Bond

H2N—R

Thermo Scientific UltraLink Biosupport binding capacity for various proteins.

Capacity Protein Coupling Buffer

35.0mg/mL Myoglobin 0.1MCHES,1.0Msodiumcitrate,pH9.0

21.5mg/mL PenicillinAcylase 0.1Msodiumphosphate,1.1Msodiumsulfate,pH7.4

20.9mg/mL a-chymotrypsin 0.1Mborate,1.5sodiumsulfate,pH9.0

35.5mg/mL BSA 0.1Mborate,1.5sodiumsulfate,pH9.0

29.8mg/mL Lysozyme 0.1Mborate,1.0sodiumsulfate,pH9.0

21.0mg/mL HumanIgG 0.1Mborate,1.5sodiumsulfate,pH9.0

Athirdoptionforimmobilizingamine-containingaffinityligandsistheuseofcarbonyldiimidazole(CDI)toactivatehydroxylsonagarosesupportstoformreactiveimidazolecarbamates.Thisreactivegroupisformedonthesupportinorganicsolventandstoredasasuspensioninacetonetopreventhydrolysis.Reactionofthesupportinanaqueouscouplingbufferwithprimaryamine-containingligandscauseslossoftheimidazolegroupsandformationofcarbamatelinkages.ThecouplingprocessoccursatbasicpH(8.5–10),butitisaslowerreactionwithproteinsthanreductiveaminationorazlactonecoupling.ThermoScientificPierceCDISupportsareavailablewiththeCDI-activatedgroup,andtheyareparticularlyadeptatimmobilizingpeptidesandsmallorganicmolecules.Thereactionalsocanbedoneinorganicsolventtopermitcouplingofwater-insolubleligands.

Reactive Imidazole Carbamate on Thermo Scientific Pierce

CDI SupportsAffinity Ligand Coupled

via Carbamate Bond

O

NH

O

RNO O NH2N—R

NHS-ActivatedAgaroseresinusessafe,reliableNHS-esterchemistryanddoesnotrequirehazardouschemicalsforimmobilization.Otheramine-reactivesupports,suchasperiodate-oxidizedresins,usetoxicsodiumcyanoborohydridetostabilizethereactionlinkagetoprimaryaminesandtake4to6hourstocomplete.Traditionalmethodssuchascyanogenbromide-activatedsupportsalsocoupleamines;however,thischemistryresultsinnonspecificbindingandconstantslowleakageofthecoupledligand.ThePierceNHS-ActivatedAgaroseusesthestableNHS-esterchemistrythatallowsimmobilizationreactionstobecompletedinlessthan1hour.

TheNHS-ActivatedAgarosecouplingreactionisperformedinanamine-freebufferatpH7-9.Proteincouplingefficiencyistypicallygreaterthan80%,regardlessoftheligand’smolecularweightorpI.Oncealigandisimmobilized,thepreparedresincanbeusedformultipleaffinitypurificationprocedures.Thecrosslinkedbeadedagarosehasfastlinearflowpotential,makingitusefulforgravity-flowandlow-tomedium-pressureapplications.


NHS-ActivatedAgaroseresinisavailableinananhydrousacetoneslurryandasdrypowder.Theuniquedryformdoesnotrequirestorageinorremovalanddisposaloftheacetonesolvent.Inaddition,thedryresinisidealforcouplingreactionswithdilutesamplesbecauseitconcentratesthesampleastheresinswells,reducingthevolumeofthestartingmaterialandresultinginhighlyefficientligandimmobilization.

O NH

O

O

O

O

O

N

NH2 –R

O NH

O

O

R

HN

Coupling Affinity Ligands through Sulfhydryl Groups

Itisoftenadvantageoustoimmobilizeaffinityligandsthroughfunctionalgroupsotherthanjustamines.Inparticular,thethiolgroupcanbeusedtodirectcouplingreactionsawayfromactivecentersorbindingsitesoncertainproteinmolecules.Becauseaminesoccuratmanypositionsonaprotein’ssurface,itisusuallydifficulttopredictwhereanamine-targetedcouplingreactionwilloccur.However,ifsulfhydrylgroupsthattypicallyarepresentinfewernumbersaretargetedforimmobilization,thencouplingcanbedoneatdiscretesitesinaproteinorpeptide.Thiolgroups(sulfhydryls)maybeindigenouswithinaproteinmoleculeortheycanbeaddedthroughthereductionofdisulfidesorthroughtheuseofvariousthiolationreagents.Sulfhydrylsalsocanbeaddedtopeptideaffinityligandsatthetimeofpeptidesynthesisbyaddingacysteineresidueatoneendofthemolecule.Thisensuresthateverypeptidemoleculewillbeorientedonthesupportinthesamewayafterimmobilization.

ThermoScientificSulfoLinkCouplingResinisdesignedtoefficientlyreactwiththiol-containingmoleculesandimmobilizethemthroughathioetherlinkage.Thesupportcontainsaniodo-acetylgroupattheendofalongspacerarm,whichreactswithsulfhydrylsthroughdisplacementoftheiodine.OptimalconditionsforthereactionareanaqueousenvironmentatslightlybasicpH,whereinaminesarenotveryreactivetowardtheiodoacetylfunction,butthiolsarehighlyreactiveduetotheirincreasednucleophilicity.Thethioetherbondthatisformedprovidesastablelinkagetoanysulfhydryl-containingmolecule.

HS R

Reactive Iodoacetyl Groupon Thermo ScientificSulfoLink Supports

Affinity Ligand Coupled via

Thioether Bond

HN

O

SR

HN

O

I

Coupling Affinity Ligands through Carbonyl Groups

Mostbiologicalmoleculesdonotcontaincarbonylketonesoraldehydesintheirnativestate.However,itmightbeusefultocreatesuchgroupsonproteinstoformasiteforimmobilizationthatdirectscovalentcouplingawayfromactivecentersorbindingsites.Glycoconjugates,suchasglycoproteinsorglycolipids,usuallycontainsugarresiduesthathavehydroxylsonadjacentcarbonatoms,whichcanbeperiodate-oxidizedtocreatealdehydes.Controlledoxidationusing1mmsodiummeta-periodateat0°Cwillselectivelyoxidizesialicacidgroupstoformanaldehydefunctionalityoneachsugar.Usinghigherconcentrationsofperiodate(10mm)atroomtemperaturewillresultinoxidationofothersugardiolstocreateadditionalformylgroups.Aldehydesonthecarbohydrateportionofglycoconjugatescanbecovalentlylinkedwithaffinitysupportsthroughanimmobilizedhydrazide,hydrazineoraminegroupbySchiffbaseformationorreductiveamination.

ThermoScientificCarboLinkCouplingResincontainslongspacerarmsthatterminateinhydrazidegroups.Reactionofthehydrazideswithaldehydesformshydrazonelinkages,whichareaformofSchiffbasedisplayingbetterstabilitythanthoseformedbetweenanamineandanaldehyde.TheCarboLink™Resincanbeusedtoimmobilizeglycoproteins,suchasantibodies,afterperiodateoxidationofthecarbohydrate.CouplingantibodiesinthismannerspecificallytargetstheheavychainsintheFcportionofthemolecule.Sincethisisawayfromtheantigen-bindingsitesattheendoftheFvregions,immobilizationusingthisrouteoftenresultsinthebestretentionofantigen-bindingactivity.

NH

O

NH2

NH

O

N R

Reactive Hydrazide Group on Thermo Scientific

CarboLink Supports

Affinity LigandCoupled via

Hydrazone Bond

R H

O



TheCarboLinkResinalsocanbeusedtocouplecarbohydratesandsugarsthroughtheirreducingends.Aldehyde-orketone-containingsugarswillreactwiththeimmobilizedhydrazidegroupswithoutoxidationofothersugarhydroxyls.However,thisreactionmaybedramaticallyslowerthancouplingwithoxidizedsugarsbecausethesenativealdehydesorketonesareusuallytiedupinacetalorketalringstructures.Theseringscanopeninaqueoussolutiontorevealthealdehydeorketone,buttheopenstructureispresentonlyasmallpercentageofthetime.Thus,thereducingendsofsugarshavedecreasedreactivitytowardanimmobilizedhydrazide,sometimesrequiringdaysofreactiontimetoobtainacceptableimmobilizationyields.

Althoughthehydrazonebondcreatedbetweentheimmobilizedhydrazideandanaldehydeismuchmorestablethanamine-aldehydeSchiffbases,toobtainaleach-resistantlinkageitisrecommendedthattheSchiffbasebereducedwithsodiumcyanoborohydride.Thisisespeciallytrueifaligandiscoupledthathasonlyasinglepointofattachmenttothesupport.Reductionofthehydrazonecreatesastablebondthatwillperformwellinaffinitychromatographyapplications.

Affinity LigandCoupled via

Hydrazone Bond

Stable LigandLinkage

NH

O

N R

NH

OH

RN

NaCNBH3

Coupling Affinity Ligands through Carboxyl Groups

Thecarboxylgroupisafrequentconstituentofmanybiologicalmolecules.Particularly,proteinsandpeptidestypicallycontainnumerouscarboxylicacidsduetothepresenceofglutamicacid,asparticacidandtheC-terminala-carboxylategroup.Carboxylicacidsmaybeusedtoimmobilizebiologicalmoleculesthroughtheuseofacarbodiimide-mediatedreaction.Althoughnoactivatedsupportcontainsareactivegroupthatisspontaneouslyreactivewithcarboxylates,chromatographysupportscontainingamines(orhydrazides)maybeusedtoformamidebondswithcarboxyl-ates.Moleculescontainingcarboxylatesmaybeactivatedtoreactwithanimmobilizedamine(orhydrazide)throughreactionwiththewater-solublecarbodiimideEDC.

EDCreactswithcarboxylatestoformanintermediateesterthatisreactivewithnucleophilessuchasprimaryamines.ThereactiontakesplaceefficientlybetweenaboutpH4.5andpH7.5,anditiscompletewithintwotofourhours,dependingonthetemperature.Theintermediateesterissubjecttohydrolysis;therefore,itisbeneficialiftheamine-containingligandtobeimmobilizedisincludedinthereactionmediumuponadditionofEDC,soitcanreactimmediatelywiththeesterasitforms.

ThermoScientificCarboxyLinkCouplingResinortheUltraLinkDADPAResinmaybeusedtoimmobilizecarboxylate-containingligandsbyEDC.CarboxyLink®ResincontainsanineatomspacerarmandUltraLinkDADPAcontainsa12-atomspacerarmtomini-mizesterichindrance.

R

OH

O

Carboxylate-ContainingAffinity Ligand

+

EDC

Immobilized DADPA on Thermo Scientific CarboxyLink Resin

Immobilized Ligand viaAmide Bond Formation

HN

HN

HN

O

R

HN

HN NH2


Products for Immobilizing Ligands through Primary Amines

Thermo Scientific Pierce NHS Ester-Activated AgarosePierceNHS-ActivatedAgaroseResin,availableasaslurryordrypowder,allowsforthesimpleandefficientimmobilizationofproteinstoabeaded-agarosesupport.TheactivatedagarosecontainsN-hydroxysuccinimide(NHS)esterwithaspacerarmofatleast10atomsinlengththatreactswithprimaryaminesform-ingstableamidelinkages.TheNHS-ActivatedAgarosecouplingreactionisperformedinanamine-freebufferatpH7-9,withtypi-calcouplingefficienciesofmorethan85%.Thepreparedresincanbeusedformultipleaffinitypurificationprocedures.Thecrosslinkedbeadedagarosehasfastlinearflowpotential,makingitusefulforgravity-flowandlow-tomedium-pressureapplications.

O NH

O

O

O

O

O

N

O NH

H

O

ON

Aga

rose

Bea

dA

garo

seB

ead

Protein

+ NH2 Protein

Thermo Scientific NHS Ester-Activated Agarose Support immobilization chemistry.

Target: –NH2

Support:6%agarose

Binding capacity: >25mgprotein/mLresin

Time: 30minutes


Description

Pkg. Size

U.S. Price

26196 Pierce NHS-Activated Agarose, Dry Swellvolume:6-7.5mL/gofdryresin

1g $ 65

26197 Pierce NHS-Activated Agarose, Dry Swellvolume:6-7.5mL/gofdryresin

5g $200

26198 Pierce NHS-Activated Agarose Spin Columns, 0.2mL 25spincolumnscontaining33mgofNHS-ActivatedAgaroseSwellvolume:6-7.5uL/mgofdryresin

25columns $125

26199 Pierce NHS-Activated Agarose Spin Columns, 2mL 5spincolumnscontaining330mgofNHS-ActivatedAgaroseSwellvolume:6-7.5uL/mgofdryresin

5columns $125

26200 Pierce NHS-Activated Agarose Slurry 25mLofsettledresininanhydrousacetone

25mL $195



Thermo Scientific AminoLink Plus Immobilization Kits and Coupling ResinThe simplest and surest method for making an affinity purification resin with antibodies or other proteins.

AminoLinkPlusCouplingResinandImmobilizationKitsuseactivatedbeadedagaroseandarobustcouplingchemistrytoimmobilizeproteinsandotherligandsthroughprimaryamines(–NH2)totheresin.Onceanantibodyorotherligandisimmobilized,thepreparedaffinityresincanbeusedforavarietyofpurificationmethodsinvolvingbatchorcolumnchromatography.Theresinandlinkagearestableinbindingandelutionconditionstypicallyusedinaffinitychromatography,enablingpreparedresintobeusedforatleast10roundsofaffinitypurification.

TheAminoLinkPlusCouplingReactioninvolvesspontaneousformationofSchiffbasebondsbetweenaldehydes(onthesupport)andamines(ontheligand)andtheirsubsequentstabilizationbyincubationwithamildreductant(sodiumcyanoborohydride;seemoredetailedreactionschemeonnextpage).Theentirecouplingreaction,calledreductiveamination,occursinfourtosixhoursinsimplenon-aminebufferssuchasPBS.Couplingefficiencywithantibodiesandtypicalproteinsisgenerallygreaterthan85%,resultingin1to20mgofimmobilizedproteinpermilliliterofagaroseresin.

Highlights:•AminoLink Plus Coupling Resin–aldehyde-activatedcrosslinked 4%beadedagarose• Ideal for antibodies and other proteins–immobilizemolecules viaprimaryamines(–NH2)•Flexible coupling conditions–efficient(>85%)couplingover awiderangeofpH(4–10)andbufferconditions(PBSorother non-aminebufferwithorwithoutorganicsolvent);regular (PBS,pH7.2)andenhanced(borate,pH10)coupling protocolsprovided• Stable, permanent immobilization –couplingreactionresults

instable,leak-resistantsecondaryaminebondbetweenresinandligand

•Better than immobilization to CNBr-activated agarose–bondis morestableanduncharged,resultinginlessnonspecificbinding inaffinitypurificationprocedures•Versatile and reusable–preparedaffinityresinisadaptableto columnandbatchaffinitytechniquesandtheresinisreusable fortypicalapplicationsbasedonproteinbindinginteractions•Convenient kits and product sizes–chooseone-orfive-column kitcontainingcompletesetsofbuffers,reagentsandversatile spin/dripcolumns,orselectbulkresin.Bulkquantitiesare availableformanufacturingapplications

Efficient immobilization of antibodies and other proteins. Percent coupling efficiency of different proteins to 2mL of Thermo Scientific AminoLink Plus Coupling Resin using the pH 7.2 coupling protocol.

ProteinProtein Applied

(mg/mL)Protein Coupled

(mg/mL)

Percent Coupled

ProteinG 4.6 4.0 83

MouseIgG 4.7 4.5 96

RatIgG 4.7 4.4 93

GoatIgG 0.9 0.8 84

HumanIgG 4.8 4.6 97

HumanIgM 0.9 0.8 93

Aga

rose

Bea

d

C

O

H

+ NH2 Protein

Aga

rose

Bea

d

C

O

NH

Protein

Thermo Scientific AminoLink Support immobilization chemistry.

Target: –NH2

Support:Highlycrosslinked4%agarose

Binding capacity:20mgprotein/mLresin

Time: 4hours

ReferencesBeall,A.,et al.(1999).J. Biol. Chem.274(16),11344–11351.Nakasato,Y.R.,et al.(1999).Clin. Chem. 45,2150–2157.Allan,B.B.,et al.(2000).Science289,444–448.Lu,R.,et al.(2000).J. Neurochem.74,320–326


Description

Pkg. Size

U.S. Price

44894 AminoLink Plus Immobilization Kit Sufficient reagents for preparing five affinity columns. Includes:AminoLinkColumn

PhosphateBufferedSaline(PBS)Citrate-CarbonateBufferQuenchingBufferWashSolutionSodiumCyanoborohydrideSolution(5M)ColumnAccessories

Kit

5x2mL1pack1pack50mL240mL0.5mL

$385

20394 AminoLink Plus Immobilization Trial Kit Sufficient reagents for preparing one affinity column. Includes:AminoLinkColumn

PhosphateBufferedSaline(PBS)Citrate-CarbonateBufferQuenchingBufferWashSolutionSodiumCyanoborohydrideSolution(5M)ColumnAccessories

Kit

1x2mL1pack1pack15mL50mL0.5mL

$105

20475 AminoLink Plus Micro Immobilization Kit Sufficient reagents for 10 coupling reactions using 25–100µg of protein and 20 affinity purifications.Includes:AminoLinkPlusSpinColumns, eachcontaining400μLof25%slurry PhosphateBufferedSaline QuenchingBuffer SodiumCyanoborohydrideSolution WashSolution ElutionBuffer MicrocentrifugeCollectionTubes

Kit10each1pack60mL0.5mL25mL50mL200each

$301

20501 AminoLink Plus Coupling Resin 10mL $105

20505 AminoLink Plus Coupling Resin 50mL $360


Thermo Scientific AminoLink Immobilization Kits and Coupling ResinLinks with primary amines (lysine residues and N-terminus) on proteins, peptides, antigens or antibodies.

AminoLinkCouplingResiniscrosslinked4%beadedagarosethathasbeenactivatedwithaldehydegroups.Proteinsandothermoleculeswithprimaryaminescanbecovalentlyattached(immobilized)toAminoLinkResintomakechromatographycolumnsforuseinaffinitypurification.Thealdehydegroupsformstablesecondaryaminebondswithprimaryaminessuchasexistinthesidechainoflysine(K)residues,whicharegenerallyabundantandreadilyaccessibleinproteins.Onceaproteinisimmobilized,thepreparedaffinityresincanbeusedforavarietyofbatchandcolumnaffinitypurificationmethodsinvolvingbindinginteractionswiththeimmobilizedprotein.Theresinandlinkagearestableinmostbindingandelutionconditionstypicallyusedinaffinitychromatography,enablingpreparedresintobeusedformultipleroundsofaffinitypurificationprocedures.

Highlights:•AminoLink Coupling Resin–aldehyde-activatedcrosslinked 4%beadedagarose• Ideal for antibodies and other proteins–immobilizemolecules viaprimaryamines(–NH2)•Flexible coupling conditions–efficient(>85%)couplingovera widerangeofpH(4–10)andbufferconditions(PBSorother non-aminebufferwithorwithoutorganicsolvent)•Stable, permanent immobilization–couplingreactionresultsin stable,leak-resistantsecondaryaminebondbetweenresin andligand•Better than immobilization to CNBr-activated agarose–bondis morestableanduncharged,resultinginlessnonspecificbinding inaffinitypurificationprocedures•Versatile and reusable–preparedaffinityresinisadaptableto columnandbatchaffinitytechniquesandtheresinisreusable fortypicalapplicationsbasedonproteinbindinginteractions

Aga

rose

Bea

d

C

O

H

+ NH2 Protein

Aga

rose

Bea

d

C

O

NH

Protein

Thermo Scientific AminoLink Support detailed immobilization chemistry.

Target: –NH2

Support:4%agarose

Binding capacity:1-20mgprotein/mLresin

Time: 4hours

Efficient immobilization at a variety of pH values. TheeffectofcouplingbufferpHonpercentcouplingefficiencyof9.58mgofhumanIgGto2mLofThermoScientificAminoLinkResinusingthestandardcouplingprotocol.

pH Coupling Efficiency of 9.58mg Human IgG

4 91.8%

5 92.7%

6 89.1%

7 87.3%

8* 85.3%

9* 94.9%

10* 98.4%

* Schiff-base formation occurs readily at high pH, but reduction to stable secondary amine bond requires subsequent incubation with sodium cyanoborohydride (AminoLink Reductant) at pH 7.2.

ReferencesCheadle,C.,et al.(1994).J. Biol. Chem.269(39),24034–24039.Cofano,F.,et al.(1990).J. Biol. Chem.265(7),4064–4071.DeSilva,B.S.andWilson,G.S.(1995).J. Immunol. Method188,9–19.Rivero-Lezcano,O.M.,et al.(1994).J. Biol. Chem.269(26),17363–17366.Czermak,B.J.,et al.(1999).J. Immunol.162,2321–2325.Assad,F.F.,et al.(2001).J. Cell Biol.152,531–543.Zuk,P.A.andElferink,L.A.(2000).J. Biol. Chem.275(35),26754–26764.6.


Description

Pkg. Size

U.S. Price

20381 AminoLink Coupling Resin 10mL $105

20382 AminoLink Coupling Resin 50mL $380

44890 AminoLink Immobilization Kit Sufficient reagents to prepare five affinity columns. Each column can be used for up to 10 affinity purifications. Includes:AminoLinkColumns

AminoLinkCouplingBufferQuenchingBufferWashSolution,SodiumCyanoborohydrideSolution(5M)ColumnAccessories

Kit

5x2mL250mL50mL240mL0.5mL

$399

20384 AminoLink Immobilization Trial KitIncludes:AminoLinkColumnReagentsandBuffers

TrialKit1x2mL

$106

44892 AminoLink Reductant(Sodiumcyanoborohydride)

2x1g $ 39



Thermo Scientific UltraLink BiosupportA durable, polyacrylamide resin, activated for efficient coupling of proteins.

UltraLinkBiosupportisadurable,porousresinthatisactivatedtoenableefficientanddirectcovalentimmobilizationofproteinsandotherbiomoleculesthroughtheirprimaryaminesforuseinaffinitypurificationprocedures.

Highlights:•High coupling efficiency and capacity–immobilizesproteins withveryhighefficiencyandcouplingcapacityin1hour•Specific and leak-proof coupling chemistry–reactsspecifically withprimaryamines(–NH2),resultinginamidebondsthatare stableforuseinmanyaffinitypurificationprocedures;coupling reactionhasnoleavinggrouptocontaminatesamples•Easy to use–nopre-swellingorsecondaryreagentsrequired; simplyweightheneededamountofdrysupportandaddthe ligandsolutiontoinitiatecouplingreaction•Flexible coupling conditions–performimmobilizationreaction inanyofavarietyofnon-aminebuffersandpHlevels;coupling ismostefficientinbufferscontainingalyotropicsaltsuchas sodiumcitrate;couplingcompatiblewithorwithoutorganicsolvent•Excellent reusability–preparedaffinityresincanbeusedwith typicalbindingandelutionproceduresformorethan100cycles ofaffinitypurificationwithoutsignificantlossofbindingcapacity•Durable, high-performance resin–porousbeadshavea60μm diameter,canwithstand100psi(6.9bar)andallowforlinear flow-throughratesof3,000cm/hour

H2N+

AmineLigand

Protein

UtraLink Biosupport(Azlactone Ring on Bead)

Amide Bond Formationwith Ring Opening

NHO

NH

O

O

N

O

CH3

CH3

Resi

nB

ead

Resi

nB

ead

Protein

Thermo Scientific UltraLink Biosupport immobilization chemistry.

Target: –NH2

Support:Polyacrylamide/azlactonecopolymer

Binding capacity:>18mgprotein/mLresin

Time: 4hours

References1.Ju,T.,et al.(2002).J. Biol. Chem.277,169–177.2.Ju,T.,et al.(2002).J. Biol. Chem.277,178–186.3.Kornfeld,R.,et al.(1998).J. Biol. Chem. 273,23202–23210.4.Liu,L.A.andEngvall,E.(1999).J. Biol. Chem.274, 38171–38176.


Description

Pkg. Size

U.S. Price

53110 UltraLink Biosupport (8-10mL) 1.25g $155

53111 UltraLink Biosupport (50mL) 6.25g $539

28388 BupH Citrate-Carbonate Buffer Packs 10packs $ 64

28386 BupH Citrate-MOPS Buffer Packs 10packs $ 58

Thermo Scientific Pierce CDI SupportsCarbonyldiimidazole-activated resins for ligand immobilization.

Highlights:•Reliable coupling chemistry–immobilizationoccursthroughthe reactionofN-nucleophileswith1,1’-carbonyldiimidazolegroups oftheresintoformastable,unchargedN-alkylcarbamatelinkages•Easy-to-use–nosecondaryreagentsneeded;justwashequilibrate resininalkalinecouplingbufferandaddligand;reactionproceeds spontaneously•Stable activation–half-lifeofhydrolysisismuchlongerthan hydroxysuccinimideesteractivations,makingimmobilization reactionssimplertoprepareandcontrol;simplifiesfiltrationand washingbeforeaddingaligandorprotein•Well-defined coupling conditions–reactionismostefficientwith primaryaminesatpH9-11

CDI-Activated Crosslinked 6% Beaded Agarose:•Beadedagarose–themostpopularresinforroutineaffinity purificationmethods•Highlyactivated–atleast50μmol1,1’-carbonyldiimidazole(CDI) groupspermilliliterofresin•Convenientform–suppliedasstabilized,50%slurryinacetone

CDI-Activated Trisacryl® GF-2000:•Trisacrylresin–rigid,polyacrylamidematrixallowsforhighflowrates•Hydrophilicmatrixwithoutchargeeffects–providesforlow nonspecificbinding•Highlyactivated–atleast50μmol1,1’-carbonyldiimidazole(CDI) groupspermilliliterofresin•Convenientform–suppliedasstabilized,50%slurryinacetone

Resi

nB

eadO C

N

N

O

OC

HN

O

H2N

ImidazoleCarbamate

AmineLigand

CarbamateLinkage

Resi

nB

ead

Protein Protein+

CDI immobilization chemistry.

References1.Shenoy,S.K.,et al.(2001).Science294,1307–1313.2.Richardson,R.T.,et al.(2000). J. Biol. Chem.275,30378–30386.3.Tanaka,M,et al.(2005).PLoSBiology3,764–776.


Description

Pkg. Size

U.S. Price

20259 Pierce CDI (6X) Support1,1’-Carbonyldiimidazoleactivatedcrosslinked6%beadedagaroseSupplied:stabilizedinacetoneslurryagarosehydratedparticlesize:45–165μmActivationlevel:>50μmol/mLofresin

10mL $ 85

20377 Pierce CDI Trisacryl Support 50mL $442


Products for Immobilizing Ligands through Sulfhydryl Groups

Thermo Scientific SulfoLink Immobilization Kits and Coupling ResinCovalent immobilization of sulfhydryl-containing peptides or proteins for affinity purification.

SulfoLinkCouplingResinisporous,crosslinked6%beadedagarosethathasbeenactivatedwithiodoacetylgroups.Whenincubatedwithasolutionofpeptideorproteinthatcontainsreducedcysteineresidues,theiodoacetylgroupsreactspecificallyandefficientlywiththeexposedsulfhydryls(–SH)toformcovalentandirrevers-iblethioetherbondsthatpermanentlyattachthepeptideorproteintotheresin.Theresultisacustom-madeaffinityresinforpurifica-tionofantibodies,antigensandothermoleculesofinterest.

Highlights:•Specific conjugation through sulfhydryl (–SH) groups–the iodoacetylgroupsreactspecificallywithsulhydrylstoform irreversiblethioetherbonds•Separate kits optimized for peptides or proteins–kitsinclude optimizedreagentsforpreparingpeptideorproteinsamplesfor efficientimmobilization•Fast –spincolumnsincreaseprotocolspeed;prepareand couplesamplesin2hours(peptides)to3.5hours(proteins)•Flexible coupling conditions–usepH7.5–9.0aqueousbuffers, organicsolvent(e.g.,20%DMSO)ordenaturant(guanidine•HCl), asneededforproteinorpeptidesolubilityduringcouplingreaction•Easy-to-follow instructions–streamLinedprotocolsforsample preparation,immobilization,andaffinitypurification•High capacity–immobilize1–2mgpeptideor2–20mgprotein per2–mLcolumnofSulfoLinkCouplingResin

HS+HN

I

O

HN

S

O

Thermo Scientific SulfoLink Coupling Resin

ThioetherBond

12-atomSpacer

IodoacetylGroup

+ HI

Immobilized Peptide

Reduced SulfhydrylMolecule

Aga

rose

Bea

d

Peptide

Peptide

Aga

rose

Bea

d

Thermo Scientific SulfoLink Support immobilization chemistry.

Target: –SH

Support:6%agaroseorUltraLink®Resin


Time: 2-3.5hours

Peptide A Calcitonin Peptide0

25

50

75

100

+TCEP

+TCEP

-TCEP-TCEP

Perc

ent

Imm

obili

zed

Improved retention of peptides on Thermo Scientific SulfoLink Resin with TCEP.TCEPeffectivelyreducespeptidestomaximizeimmobilizationefficiency.Twopeptides(PeptideAandhumancalcitoninpeptide)wereincubatedwith25mmTCEPfor30minutesandimmobilizedontoSulfoLinkResinviatheirreducedsulfhydrylgroups.PeptideA’scysteinehadoxidizedduringlong-termstorageandthecalcitoninpeptidecontainedaninternaldisulfidebond.Eachpeptidealsocon-tainedanamine-terminalfluorescentprobebywhichthebindingofthepeptidecouldbemonitoredduringtheimmobilizationandwashsteps. ReferencesGrunwald,R.andMeissner,G.(1995).J. Biol. Chem.270(19),11338–11347.Seubert,P.,et al.(1993).Nature361,260–263.Sukegawa,J.,et al.(1995).J. Biol. Chem.270(26),15702–15706.Wisniewski,J.R.,et al. (1994).J. Biol. Chem.269(46),29261–29264.Sakaguchi,K.,et al.(2000).J. Biol. Chem.275,9278–9283.Tan,M.,et al.(2000).Proc. Natl. Acad. Sci. USA97,109–114.Quill,T.A.,et al.(2001).Proc. Natl. Acad. Sci. USA98,12527–12531.Tokumaru,H.,et al.(2001).Cell 104,421–432.Assad,F.F.,et al.(2001).J. Cell Biol.152,531–543.


Description

Pkg. Size

U.S. Price

44995 SulfoLink Immobilization Kit for Proteins Sufficient reagents to prepare five affinity columns. Kitcontains:SulfoLinkColumns

SulfoLinkSamplePreparationBufferSulfoLinkCouplingBufferWashSolution2-Mercaptoethylamine•HClL-Cysteine•HClZebaDesaltSpinColumnsPhosphateBufferedSalineColumnsAccessories

Kit

5x2mL7.5mL500mL120mL5x6mg100mg5x5mL1Pack

$ 399

44999 SulfoLink Immobilization Kit for Peptides Sufficient reagents to prepare five affinity columns. Kitcontains:SulfoLinkColumns

SulfoLinkCouplingBufferWashSolutionBond-BreakerTCEPSolutionL-Cysteine•HClPhosphateBufferedSalineColumnAccessories

Kit

5x2mL120mL120mL0.5mL100mg1Pack

$ 399

20325 SulfoLink Immobilization Trial Kit Sufficient reagents to prepare 1 affinity column with a peptide or protein. Kitcontains:SulfoLinkColumn

SulfoLinkSamplePreparationBufferSulfoLinkCouplingBufferWashSolution2-Mercaptoethylamine•HClBond-BreakerTCEPSolutionCysteine•HClZebaDesaltSpinColumnPhosphateBufferedSalineColumnsAccessories

Kit

1x2mL7.5mL120mL25mL6mg0.5mL100mg5mL1Pack

$ 170

20401 SulfoLink Coupling Resin 10mL $ 13520402 SulfoLink Coupling Resin 50mL $ 47420404 SulfoLink Coupling Resin 250mL $1900



Thermo Scientific UltraLink Iodoacetyl ResinPolyacrylamide resin for coupling sulfhydryl-containing ligands.

UltraLinkIodoacetylResinisadurable,porousresinthatisactivatedtoenableefficientanddirectcovalentimmoblizationofpeptidesandotherligandsthroughtheirsulfhydrylgroups(–SH)foruseinaffinitypurificationprocedures.Thebeadedresinisahydrophiliccopolymerofpolyacrylamideandazlactonehavingarigidpoly-mericstructurewithhighsurfaceareaandporevolume.Eachazlactonegrouphasbeenmodifiedtoforma15-atomspacerarmthatterminatesinaniodoacetylgroup,whichiscapableofreact-ingwithsulfhydrylgroups(e.g.,sidechainofreducedcysteineresidues)tocovalentlyimmobilizepeptideorothersulfhydryl-containingligands.ThebeadstructureandefficientlycouplingchemistryofIodoacetyl-ActivatedUltraLinkSupportresultsinhighproteinbindingcapacity,highlinearflowrates,lownonspecificbindingandoverallsuperiorperformanceinaffinitychromatography.UltraLinkResinsareidealformediumpressureapplicationssuchasFPLC.

Highlights:•High coupling efficiency and capacity–immobilizessulfhydryl- containingproteinsorotherligandswithhighefficiencyand couplingcapacityin1hour•Specific and leak-proof coupling chemistry–reactsspecifically withsulfhydrylgroups(reducedthiols),resultinginthioether bondsthatarestableforuseinmanyaffinitypurificationprocedures•Simple coupling conditions–performimmobilizationreactionin anyofavarietyofbuffers;couplingismostefficientandspecific atpH8.0–8.5;couplingcompatiblewithorwithoutorganicsolvent•Excellent reusability–preparedaffinityresincanbeusedwith typicalbindingandelutionproceduresformanycyclesofaffnity purificationwithoutsignificantlossofbindingcapacity•Durable, high-performance resin–porousbeadshavea60μm diameter,canwithstand100psi(6.9bar)andallowforlinear flow-throughratesof3,000cm/hour

References1.Ju,T.,et al.(2002).J. Biol. Chem.277,169–177.2.Hill,K.,et al.(2000).J. Biol. Chem.275(6),3741–3744.3.Liu,L.A.andEngvall,E.(1999). J. Biol. Chem.274,38171–38176.4.Bicknell,A.B.,et al.(2001).Cell105,903–912.


Description

Pkg. Size

U.S. Price

53155 UltraLink Iodoacetyl ResinSupport:UltraLinkBiosupport

10mL $188

HS+HN

IO

HN

SO

Iodoacetyl-Activated Thermo Scientific UltraLink Resin

Thioether

Bond15-atomSpacer

IodoacetylGroup

+ HI

Immobilized PeptideReduced SulfhydrylMolecule

Ther

mo

Scie

ntifi

c U

ltraL

ink

Bea

d

Ther

mo

Scie

ntifi

c U

ltraL

ink

Bea

d

Peptide Peptide

Thermo Scientific UtraLink Iodoacetyl immobilization chemistry.

Thermo Scientific UltraLink Iodoacetyl Micro Peptide Coupling KitEasily prepare a small-scale affinity column with sulfhydryl- containing peptides.

TheMicroPeptideCouplingKitisamicrocentrifugespincolumnkitforimmobilizingsmallamounts(25–250μg)ofsulfhydryl-containingpeptides(e.g.,cysteine-terminatedpeptides)ontoabeadedporousresintocreateasmall,reusableaffinitycolumn.Thecouplingandaffinitypurificationproceduresareoptimizedforsmallsamplevolumes(200–300μL).Washandelutionstepsareachievedrapidlyandefficientlywiththeconvenientmicrocentrifugespincolumns.Eachkitcontainssufficientreagentsfor10couplingreactionsand20affinitypurifications.Thekitisidealforimmobilizingpeptideantigensthatcontainingaterminalcysteineresidueforuseinpurifyingspecificantibodiesfromsmallserum,ascitesorculturesupernatantsamples.

Highlights:•Optimized for small scale–idealforcouplingsmallamounts (25–250μg)ofsulfhydryl-containingpeptideorproteinfor purificationofspecificantibodiesfromcrudeserum•High-performance affinity resin–usesdurable,polyacrylamide- basedUltraLinkIodoacetylResinforspecificreactionto sulfhydrylgroups•Efficient coupling chemistry–immobilizationefficiency>85% (asmeasuredwith1hourreactionusinginsulin,calcitoninand osteocalcinpeptides)•Fast and easy to use–performwashandelutionstepsusing amicrocentrifuge•Reusable–usepreparedpeptideresinseveraltimeswithno significantlossofcapacity


Description

Pkg. Size

U.S. Price

20485 Micro Peptide Coupling Kit Sufficient for coupling 10 sulfhydryl-containing peptides or proteins and perform 20 affinity purifications. Kitcontents:UltraLinkIodoacetylSpinColumns,0.1mL

CouplingBufferL-Cysteine-HClWashSolutionPBSPack(makes500mL)IgGElutionBufferCollectionTubes

Kit

10columns100mL100mg25mL1pack50mL200tubes

$357


Thermo Scientific Disulfide Reducing AgentsReduce disulfide bonds to produce sulfhydryl groups for immobilization on SulfoLink or UltraLink Resins.

Freesulfhydrylsarerequiredforimmobilizationontosulfhydryl-reactiveaffinitysupports.Cysteinesinproteinsandpeptidesusuallyexistascystines(disulfidebridges)andmustbereducedtoexposesulfhydrylsforcoupling.Reductioncanbeaccomplishedwithfreeorimmobilizedreducingagents.Freereducingagentsareefficientinreducingalldisulfidesinproteins,includingthoseburiedinthetertiarystructure,buttheymustberemovedfromthereducedsamplewithadesaltingcolumnbeforecouplingtothesupport.Immobilizedreducingagentsenablereductionofdisulfidesandsimpleremovalofthereducedsamplefromthereducingagent.Thisisespeciallyhelpfulwhenreducingpeptideswhosesmallsizepreventsthemfrombeingeffectivelydesalted.

PHO

O

OHO

OH

O

Cl–

TCEP•HClMW 286.65

H+

HSNH3 Cl–

2-Mercaptoethylamine•HCIMW 113.61

SHHS

OH

OH

DTTMW 154.25

+


Description

Pkg. Size

U.S. Price

20408 2-Mercaptoethylamine•HCl 6x6mg $135

20290 DTT, Cleland’s Reagent(Dithiothreitol)

5g $105

20291 Dithiothreitol (DTT) in No-Weigh™ Format7.7mgDTT/tube.Makes100μLof0.5MDTT.

48tubes $125

20490 TCEP•HCl(Tris[2-carboxyethyl]phosphinehydrochloride)

1g $ 50

20491 TCEP•HCl 10g $299

77720 Bond-Breaker TCEP Solution, Neutral pH 5mL $108

77712 Immobilized TCEP Disulfide Reducing Gel 5mL $118

Thermo Scientific Ellman’s Reagent and Sulfhydryl Addition KitMeasure and add free sulfhydryls to ensure success of cysteine-targeted immobilization.

Ellman’sReagent,alsocalledDTNB,isaversatilewater-solublecompoundforquantifyingfreesulfhydrylgroupsinsolution.Asolutionofthiscompoundproducesameasurableyellow-coloredproductwhenitreactswithsulfhydryls(–SHgroups).Bytestinganunknownsample,suchasapeptidehavingaterminalcysteineresidue,comparedtoastandardcurvemadewithknownamountsoffree,reducedcysteine(Product#44889),availabilityofreducedsulfhydrylsinthesamplecanbedetermined.

TheSulfhydrylAdditionKitprovidestheessentialreagentsandprocedureforcreatingnewsulfhydrylgroupsonaproteinorothermoleculethatcontainsavailableprimaryamines(–NH2).ThekitusesSATAreagent,whichformscovalentbondstoprimaryamines.Theresultisadditionofastable(capped)sulfhydrylgroup,whichcanlaterbeexposedbygentletreatmentwithhydroxylamine,makingthemoleculereadyforconjugationtoSulfoLinkCouplingResin,UltraLinkIodoacetylResinorothersulfhydryl-reactiveimmobilizationmethod.


Description

Pkg. Size

U.S. Price

23460 Sulfhydryl Addition KitAdds free sulfhydryl groups to proteins.Includes:SATA Hydroxylamine•HCl 10XConjugationBufferStock PhosphateBufferedSalinePack Dimethylformamide(DMF) DextranDesaltingColumn ColumnExtender Ellman’sReagent(DTNB) Cysteine•HClH2O

Kit2mg5mg20mL1pack1mL1x5mL12mg20mg

$223

22582 Ellman’s Reagent(5,5’-Dithio-bis-[2-nitrobenzoicacid])

5g $ 74

44889 Cysteine•HCl 5g $ 45



Products for Immobilizing Ligands through Carbonyl Groups

Thermo Scientific CarboLink Immobilization Kit and Coupling ResinsImmobilize polyclonal antibodies and other glycoproteins through carbohydrate groups.

CarboLinkCouplingResinandKitsprovideforcovalentimmobiliza-tionofglycoproteinsandothercarbohydrate-containingmoleculestobeadedagarose(orpolyacrylamideUltraLinkSupport)foruseinaffinitypurificationprocedures.Carbohydratemoietiesinglyco-proteinscontaincommonsugarswhosecis-diolgroupsareeasilyoxidizedwithsodiummeta-periodate(includedintheCarboLinkKit)toyieldaldehydes.WhenincubatedwiththeCarboLinkResin,thesealdehydegroupsreactspontaneouslywiththehydrazidegroupoftheactivatedresintoformstable,covalentbonds.Theimmobilizationstrategyisespeciallyusefulforglycoproteins,suchaspolyclonalantibodies,becauseitallowsattachmentofthemoleculeatdomainsthatwillnotinterferewithbindingsitesthatarecriticalfortheintendedaffinitypurification.Onceamoleculeiscoupled,thepreparedaffinityresincanbeusedmultipletimesintypicalproteinaffinitypurificationprocedures.

Highlights:•CarboLink Coupling Resin–hydrazide-activatedcrosslinked 6%beadedagarose(orhydrazide-activatedUltraLinkSupport, abeaded,polyacrylamideresin)•Efficient immobilization–couple1–5mgofoxidizedpolyclonal antibodyorotherglycoproteinpermilliliterofresin(CarboLink Resincontainsgreaterthan14μmolhydrazidegroupspermilliliter)•Stable linkage–resonancestructureofthehydrazonebonds aresufficientlystabletoallowmultipleroundsofaffinity purificationwithonebatchofpreparedresin;nostabilizing reductantrequired•Flexible and gentle coupling conditions–immobilizationreaction completedinsimplebuffers(PBSorothernon-aminebufferwith orwithoutorganicsolvent)atnear-neutralpH• Ideal for polyclonal antibodies–immobilizesIgGthrough carbohydratesintheFcregion,sobothantigenbindingsitesare freetointeractwiththeantigeninthemobilephase•Effective for any molecule with oxidizable sugars–firststepis oxidationofthesugargroups,whichallowsthecis-diolsofthe IgGtobetransformedintoreactivealdehydemoieties;these aldehydesthencombinewithhydrazidegroupsonthematrixto formstable,leak-resistantlinkages•Convenient kits and product sizes–chooseone-orfive-column kitcontainingcompletesetsofbuffers,oxidizingreagentand versatilespin/dripcolumnscontainingthebeadedagaroseresin; orchoosethepolyacrylamide-basedUltraLinkSupport.Bulk quantitiesareavailableformanufacturingapplications

Aga

rose

Bea

d

Aga

rose

Bea

d

NH

NH2

O

NH

NO

Thermo Scientific CarboLink Resin

23-atomSpacer

HydrazideGroup

Antibody with CarbohydrateGroups Oxidized to Form

Aldehyde Groups

Immobilized Glycoprotein

Oxidized Glycoprotein

HydrazoneBond

H

O

Thermo Scientific CarboLink Support immobilization chemistry.

Target: –CHO

Support:6%agarose


Time: 8hours

References1.Kumar,P.G.,et al.(2001).J. Biol. Chem.276,41357–41364.2.Strakova,Z.,et al.(1997).Mol. Pharmacol.51,217–224.3.Brown,M.A.,et al.(2000).J. Biol. Chem.275,19795–19802.4.Butko,P.,et al.(1999).J. Immunol.163,2761–2768.5.Sequra,M.,et al.(1999). Infect. Immun.67(9),4646


Description

Pkg. Size

U.S. Price

20355 CarboLink Immobilization Trial KitKitcontains:CarboLinkColumn

CarboLinkCouplingBufferCarboLinkWashSolutionSodiummetaperiodateZebaDesaltingColumn

Kit1x2mL60mL15mL1x5mg1x5mL

$155

44910 CarboLink Immobilization KitKitcontains:CarboLinkColumns

CarboLinkCouplingBufferCarboLinkWashSolutionSodiummetaperiodateZebaDesaltingColumns

Kit5x2mL250mL100mL5x5mg5x5mL

$454

20391 CarboLink Coupling Resin 10mL $11353149 UltraLink Hydrazide Resin 10mL $18620504 Sodium meta-Periodate 25g $ 29


Products for Immobilizing Ligands through Carboxyl Groups

Thermo Scientific CarboxyLink Immobilization Kits and Coupling ResinsImmobilize peptides via carboxyl groups to create an affinity column.

CarboxyLinkCouplingResinandKitsprovideforcovalentimmobil-izationofpeptidesorothercarboxyl-containing(–COOH)moleculestoaporous,beadedresinforuseinaffinitypurificationprocedures.CarboxyLinkResiniscrosslinkedbeadedagarose(orpolyacryl-amideUltraLinkSupport)thathasbeenactivatedwithdiamino-dipropylamine(DADPA)tocontainlongspacerarms,eachwithaprimaryamineattheend.WhenincubatedwiththeresinandthecarbodiimidecrosslinkerEDC(includedintheCarboxyLinkImmobilizationKit),carboxyl-containingmoleculesbecomeper-manentlyattachedtothesupportbystableamidebonds.Onceamoleculeiscoupled,thepreparedaffinitycolumncanbeusedmultipletimesintypicalproteinaffinitypurificationprocedures.CarboxyLinkCouplingResinscanalsobeusedtoimmobilizeotherkindsofmoleculesusingalternativeamine-reactivecrosslinkingchemistries.

Highlights:•CarboxyLink Coupling Resin–DADPA-activatedcrosslinked 4%beadedagarose(orDADPA-activatedUltraLinkSupport, abeadedpolyacrylamideresin)•Efficient immobilization–couple1–2mgofpeptidepermilliliter ofresin(CarboxyLinkAgaroseResinactivatedwithgreaterthan 16μmolaminemilliliterofresin;DADPAonUltraLinkSupport activatedwithgreaterthan40μmolaminemilliliterofresin)•Stable linkage–immoblizationresultsincovalentattachmentof carboxylgroupsbyamidebonds,allowingformultipleroundsof affinitypurificationwithonebatchofpreparedresin•Flexible and gentle coupling conditions–immobilizationreaction completedinsimpleMESorothernon-amineandnon-carboxyl, near-neutralbuffer,withorwithoutorganicsolvent.• Ideal for unmodified peptides–immobilizespeptideswith highcapacityandvariousorientationswithoutsteric hindrance,allowingforeffectiveuseinaffinitypurificationof specificantibodies•Convenient kits and product sizes–choosefive-columnkitswith completesetsofbuffers,crosslinkerandversatilespin/drip columnscontainingeithertypeofresin(agaroseor polyacrylamide)orchoosestand-aloneresinforotheruses

HN

HN NH2

Immobilized DADPA(Thermo Scientific CarboxyLink Resin)

HN

HN

HN

+O

HO

O

EDC Crosslinker

Carboxyl Ligand(e.g., peptide C-terminus)

Covalently Immobilized Ligand(attached by amide bond and long spacer arm)

Aga

rose

Bea

d

Peptide

Peptide

Aga

rose

Bea

d

Thermo Scientific CarboxyLink Support immobilization chemistry.

Target: –COOH

Support:4%agaroseorUltraLink®Resin


Time: 4hours

ReferenceYoo,B.C.,et al. (2002).J. Biol. Chem.277,15325–15332.


Description

Pkg. Size

U.S. Price

44899 CarboxyLink Immobilization KitKitcontains:CarboxyLinkColumns(DADPAagarose)

EDCCrosslinkerCouplingBuffer(MES-bufferedSaline)WashSolution(1MNaCl)ColumnAccessories

Kit

5x2mL5x60mg500mL120mL

$393

20266 CarboxyLink Coupling ResinSupport:Crosslinked4%beadedagaroseLoading:16–20μmolavailableaminogroups/mLofresin

25mL $145

53154 CarboxyLink Immobilization Kit with UltraLink SupportKitcontains:DADPAUltraLinkColumns

EDCCrosslinkerCouplingBuffer(MES-bufferedSaline)WashSolution(1MNaCl)ColumnAccessories

Kit

5x2mL5x60mg500mL120mL

$432

22980 EDC 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride

5g $ 83

28390 BupH MES Buffered Saline Packs 10pack $112



Antibody immobilization: choosing the best Thermo Scientific Support.

AminoLink Plus Coupling Resin

UltraLink Biosupport

CarboLink Coupling Resins

SulfoLink Coupling Resins

Crosslink IP Kits See page 41

Monoclonal Antibodies Advantages:•Goodchoicewhen

onlysmallamountsofantibodyareavailable

•CoupleoverabroadpHrange

•Goodcouplingefficiency

Disadvantages:•ReductionofSchiff’sbase

withsodiumcyanoborohy-dridemayadverselyaffectmonoclonals

•Someantibodiesmaybecoupledthroughantigen-bindingsite

Advantages:•Goodchoiceifantibody

canwithstand1.0Msodiumcitrateorsulfate

•Highcapacity•Fast,efficientcoupling•Good,forlarge-scaleor

fast-flowapplications

Disadvantages:•Someantibodiesmaybe

coupledthroughantigen-bindingsite

•Someantibodiesmaypre-cipitateinhigh-saltbuffer

Advantages:•Correctlyorientsantibody•Antibodymustbeable

towithstandoxidationconditions

•Goodforantibodieswithlowavidityforantigen

Disadvantages:•Notallmonoclonalshave

carbohydrateaccessibleforcoupling

•Conditionsnecessaryforcouplingmayadverselyaffectsomemonoclonals

Advantages:•Goodchoicefor

antibodiesthathaveextremelyhighavidityfortheirantigen

•Allowsforgentleelutionconditions

Disadvantages:•Mustfirstreduceantibody

priortocoupling•Notgoodforantibodies

withlowaffinityfortheirantigens

Advantages:•Allowsforcorrect

orientationofantibodies•Gentlecouplingconditions•ProteinA/Gwillbind

mostantibodies

Disadvantages:•Ifpurifyingantigenfrom

serum,antibodiesmaybindtoProteinA/Gandco-purifywithantigen

•Crosslinkingresultsinsomelossofantibodyactivity

Polyclonal Antibodies Advantages:•Excellentcoupling

efficiency•Goodantigenrecovery

Disadvantages:•Someantibodiesmay

becoupledthroughantigen-bindingsite

Advantages:•Goodchoicefor

large-scaleorfast-flowapplications

•Highcapacity•Fast,efficientcoupling

Disadvantages:•Someantibodiesmay

becoupledthroughantigen-bindingsite

•Someantibodiesmayprecipitateinhigh-saltbuffer

Advantages:•Correctlyorientsantibody•Antibodymustbeable

towithstandoxidationconditions

•Goodforantibodieswithlowavidityforantigen

Disadvantages:•Conditionsnecessaryfor

couplingmayadverselyaffectsomeantibodies

Advantages:•Goodchoiceforanti-

bodiesthathaveavidityfortheirantigen

•Allowsforgentleelutionconditions

Disadvantages:•Mustfirstreduceantibody

priortocoupling•Notgoodforantibodies

withlowaffinityfortheirantigens


orientationofantibodies•Gentlecoupleconditions•ProteinA/Gwillbind

mostantibodies




High-Activity Antibodies Advantages:•Immobilizationofreduced

antibodyallowsforgentlerelutionconditions

Low-Activity Antibodies Advantages:•Correctlyorientsantibody

Disadvantages:•Conditionsnecessaryfor

couplingmayadverselyaffectsomemonoclonals


orientationofantibodies•Gentlecoupleconditions•ProteinA/Gwillbind

mostantibodies





Immunoprecipitation

The topic of co-immunoprecipitation (co-IP) is best preceded by a discussion of immunoprecipitation (IP) to help frame an under-standing of the principles involved.

IPisoneofthemostwidelyusedmethodsforantigendetectionandpurification.AnimportantcharacteristicofIPreactionsistheirpotentialtodelivernotonlythetargetprotein,butalsoothermacromoleculesthatinteractwiththetarget.

The IP PrincipleTheprincipleofanIPisverysimple(Figure1).Anantibody(monoclonalorpolyclonal)againstaspecifictargetantigenisallowedtoformanimmunecomplexwiththattargetinasample,suchasacelllysate.TheimmunecomplexisthencapturedonasolidsupporttowhicheitherProteinAorProteinGhasbeenimmobilized(ProteinAorProteinGbindstotheantibody,whichisboundtoitsantigen).Theprocessofcapturingthiscomplexfromthesolutionisreferredtoasprecipitation.Anyproteinsnot“precipitated”bytheimmobilizedProteinAorProteinGsupportarewashedaway.Finally,componentsoftheboundimmunecomplex(bothantigenandantibody)areelutedfromthesupportandanalyzedbySDS-PAGE(gelelectrophoresis),oftenfollowedbyWesternblotdetectiontoverifytheidentityoftheantigen.

Coupled Antibody

Spin and wash

Cell Lysate or Protein MixtureIncubation with

Antibody-coupled Resin

Antigen

Elute

Analyze

Figure 1. Summary of a traditional immunoprecipitation procedure.

Traditionalimmunoprecipitationinvolvesthefollowingsteps:

1.Formtheantigen-antibodycomplex(immunecomplex)by incubatingspecificantibodywiththeantigen-containingsample for1hourtoseveralhours.2.CapturetheimmunecomplextoProteinAorProteinGagarose beadsbyincubationfor0.5-2hours.3.Removeanynon-boundprotein(non-immunecomplexsample components)fromtheprecipitatedcomplexbywashingbeads withadditionalsamplebuffer.4.BoilbeadsinreducingSDS-PAGEsampleloadingbuffer.5.Recoverelutedsampleinloadingbufferandanalyzeby SDS-PAGE.6.PerformWesternblotanalysis,probingwithantigen- specificantibody.

Co-IP vs. IPCo-IPisapopulartechniqueforproteininteractiondiscovery.Co-IPisconductedinessentiallythesamemannerasIP(Figure2).However,inaco-IPthetargetantigenprecipitatedbytheantibody“co-precipitates”abindingpartner/proteincomplexfromalysate;i.e.,theinteractingproteinisboundtothetargetantigen,whichbecomesboundbytheantibodythatbecomescapturedontheProteinAorProteinGresin.Theassumptionusuallymadewhenassociatedproteinsareco-precipitatedisthattheseproteinsarerelatedtothefunctionofthetargetantigenatthecellularlevel.Thisisonlyanassumption,however,thatissubjecttofurtherverification.

Coupled AntibodySpin and wash

Cell Lysate or Protein MixtureIncubation with

Antibody-coupled Resin

Antigen

Protein Interactingwith Antigen

Elute

Analyze

Figure 2. Summary of a traditional co-immunoprecipitation procedure.

IP/Co-IP


Traditional Methods vs. Thermo Scientific Pierce Innovations for Co-IP

Problems with Traditional Co-IP MethodsThetraditionalco-IPprotocolhascertaindeficienciesrelatingtothefundamentalformatoftheassay,theantibodyandassociatedchemistry.OneofthemostcommonlyencounteredproblemswiththetraditionalIPandco-IPapproachisinterferencefromantibodybandsingelanalysis.Inthosecasesinwhichseveralproteinsmaybeco-precipitatedwiththetarget,presenceoftheco-elutedantibodyheavyandlightchains(25and50kDabandsinreducingSDS-polyacrylamidegel)inthepreparationcanobscuretheresults.Theidealsituationwouldbetoconducttheco-IPwithoutcontaminationoftheelutedantigenwithantibody.Withthispotentialinterferenceeliminated,onlytheco-precipitatedproteinswillbepresentanddetectedonagel.ThisandothershortcomingsofthetraditionalprotocolandoursolutionsaresummarizedinTable1.

Table 1. Comparison of traditional Co-IP and Thermo Scientific Pierce Co-IP Products.

Traditional Co-IP Problems Thermo Scientific Pierce Product Solutions

Batch processing of the precipitated complex in a single tube:resultsininefficientwashingofnon-boundproteinsfromthesupportandinresinlossduetodecantingwashbufferfromtubeviaapipette,whichlowersproteinyields

Spin cup or spin tube processing:dedicatedIPandco-IPkitsthatcontainspin-cuporspintubedevicesthatincreasewashingefficiencyoffermoreeffectiveelutionofantigenandassociatedproteinandeliminateresinloss,yieldingsignificantlyhigherproteinrecoveriesandmoreconsistentresults

Antibody fragment interference:co-elutionofantibodyfragmentswithantigenoftenresultsinbandsinterferingwithdetectionofanyco-precipitatedproteinsonSDS-PAGE

Antibody immobilization:chemistriesdesignedtoimmobilizetheantibodytothesupport,therebyallowingelutionofonlythetargetandanyassociatedproteinsinaco-IPcomplex

Eliminate detection of antibody used for immunoprecipitation:contaminatingantibodyfragmentsbecomedenaturedduringSDS-PAGE.Incontrast,theprimaryantibodyusedduringWesternblottingisstillnative.UseCleanBlotHRPDetectionReagent(Product#21230)toonlydetectnativeantibodyandnotcontaminants.

Antibody sacrificed:asaconsequenceofharshelutionconditions,thetargetantibodyisdestroyed;antibodylossbywayoftheprotocolcanbecostly

Antibody re-used:immobilizationchemistryandmildelutionconditionsforthetargetandassociatedproteinsallowtheimmobilizedantibodytobere-equilibratedandrecycledseveraltimesintheco-IPprotocol

Approaches to Co-IP Free of Antibody Interference

Three approaches have been incorporated into several products targeted to IP and co-IP applications.

Activated Support for Antibody Immobilization — Direct StrategyInthisapproach,anantibodyisimmobilizeddirectlythroughitssurfaceaminegroups(contributedprimarilybythesidechainepsilon-aminogroupoflysine)toahigh-capacityaldehyde-activatedbeadedagarosesupport(ThermoScientificAminoLinkPlusCouplingResin).ThesupportformsaSchiff’sbasewiththeseavailableaminesthatisreducedtoformstablesecondaryaminebondsduringtheimmobilizationprocess.Thewiderangeofcouplingconditionsthatcanbeusedwiththissupportmakeitidealformaintainingbiologicalbindingactivitycriticaltothesuccessfulexecutionofaco-IPexperiment.WhenusingtheDirectStrategy,theantibodysourceshouldbefreeofcarrierprotein,whichcanalsobeimmobilized.ProductssuchasThermoScientificMelonGelIgGPurificationKits(Product#45206)caneasilyclean-upanantibodytoremovecarrierproteins.Thedirectimmobilizationofantibodiesisnotspeciesdependent,whichallowsfortheuseonnon-traditionalantibodysourcesthatdon’tbindglobulinbindingproteins(ProteinAorProteinG).ThermoScientificPierceCo-ImmunoprecipitationKits(Product#26149)andThermoScientificPierceDirectIPKit(Product#26148)usethisdirectimmobilizationapproach.Forco-IPapplica-tions,theflexibility,simplicityanddurabilityofthedirectmethodasanantibody-couplingstrategymakesitthemethodofchoicefordeliveringresultsfreefromantibodyinterference.

Antibody Orientation and Immobilization — Indirect StrategyThisstrategytakesadvantageofthebindingcharacteristicsofthetraditionalProteinAorProteinGagarosecombinedwithchemicalcrosslinkingtocovalentlylinktheantibodytothesupport.ProteinAandProteinGbindIgGclassantibodiesthroughtheFcregionthatischaracterizedprimarilybydimerizedheavychainmodifiedbycarbohydrate.Fcregionbindingnaturallyorientstheantigen-bindingdomainsoftheantibody(Fab)awayfromthesupport,makingthemavailableforbindingtotheirrespectivetargetantigen.TheIndirectStrategyiscompatiblewithantibodysamplesthatcontaincarrierproteins,providedthecarrierproteinsarewashedawaypriortocrosslinking.Toensurethattheantibodyremainsonthesupportduringtherequisiteantigenbinding,washandelutionstepsoftheprotocol,thisboundandorientedantibodyischemi-callycrosslinkedtotheProteinAorProteinGwiththebifunctionalreagentdisuccinimidylsuberate(DSS).ThermoScientificPierceCrosslinkIPKit(Product#26147)incorporatesthisstrategy.


Use of the Streptavidin:Biotin InteractionThisdirectcouplingapproachincorporatesthebindingassociationbetweenstreptavidinandbiotin.StreptavidinimmobilizedtobeadedagaroseresinorcoatedinmicroplatewellsprovidesanalternativeIPorco-IPstrategyforobtainingresultsfreefromantibodyinter-ference.Biotinylatedantibodyisboundverystronglytoeachmatrixandisnotelutedwhenmildconditionsareusedtoreleasethetargetantigen.TheIP/co-IPisconductedbyincubatingthesamplewiththebiotinylatedantibody-loadedmatrix.Elutionofthetargetantigenandanyinteractingproteinsisperformedfreeofantibodycontamination.ThermoScientificPierceStreptavidinAgaroseResin(e.g.,Product#20347)andkitproductsthatusethissupport,aswellastheThermoScientificPierceStreptavidinCoatedPlateIPKit(Product#45360),providehigh-capacitybiotin-bindingmatricessuitableforIPandco-IPapplications.

Optimization Parameters in IP and Co-IP

Classical Immune Complex Formation vs. Pre-Binding of AntibodyAchangeinprotocolfromtheclassicalimmunecomplexprecipitationisnecessarywhenusingimmobilizedantibodyintheco-IPmethod.Inthetraditionalco-IPprotocol,theimmunecomplex(antigen:antibody)isformedinsolutionbefore“precipitating”itwiththeimmobilizedProteinAorProteinGmatrix.Whenusingimmobilizedantibody,theimmunecomplexisformeddirectlyontheantibody-coupledmatrixbyincubationoftheantigen-containingsamplewiththematrix.Formationoftheimmunecomplex(thetargetantigenandanytarget-associatedprotein)anditsprecipitationoccursinonestep.

Intheimmobilizedformat,theantibodyisallowedtoincubatewiththelysate.Thematrixiswashedusingaspincupformatandtheboundproteinelutedforanalysis.Thetargetantigenandco-IPcomplexisrecoveredfreeofantibodyorantibodyfragmentcontamination,andtheantibodyisretainedinanactiveformonthesupporttobeusedinanotherco-IPcycle.

Ourresearchindicatesthatpre-bindingor-couplingtheantibodytothesupportmatrixconsistentlyresultsinthecaptureofmoretargetantigen,evenincoatedplateIPproceduresthatdonotrequireit.ThisapproachisrecommendedfortheThermoScientificPierceCoatedPlateIPKitsthatuse96-wellmicroplatescoatedwithstreptavidinorProteinA/G(Products#45360,45350,respectively).Antibodyisboundtotheplatewellspriortotheprescribedincubationwithalysatesample.Unboundproteiniseasilywashedfromthewellspriortotheelutionofthetargetandanyco-precipitatedproteins.

Evaluating a Co-IP-Captured Interaction

Intheirreviewofproteininteractions,PhizickyandFields(seeReferenceslistedbelow)presentadiscussionoftheissuestoconsiderinvalidatingasuspectedinteractionobtainedbyaco-IPexperiment.Ultimately,thefollowingquestionmustbeanswered:Doestheinteractiondetectedbyco-IPoccurin vivo,andwhatsignificancedoesithaveatthecellularlevel?AsummaryofthePhizickyandFieldsapproachtoverificationofco-IPdatafollows.

Confirm that the co-precipitated protein is obtained only by antibody against the targetUsemonoclonalantibodiesintheco-IPprotocol.Whenonlyapolyclonalantibodyisavailable,pre-treatmentoftheantibodywithsampledevoidoftheprimarytarget(baitprotein)mayberequiredtoassurethatthepolyclonalantibodydoesnotcontainclonesorcontaminantsthatbindpreyprotein(s)directly.Pre-adsorptiontoextractsdevoidoftargetorpre-purificationofpolyclonalIPantibodiesagainstanaffinitycolumncontainingpuretargetantigensafeguardsagainstafalse-positiveco-IP.

Conclude that antibody against the target antigen does not itself recognize the co-precipitated protein(s)Useindependentlyderivedantibodiesthathavedemonstratedspecificitiesagainstdifferentepitopesonthetargetprotein.Theiruseservesasverificationthatthetarget(bait)-directedantibodieshavenoaffinityforthetarget-associatedpreyproteinsrecoveredduringtheco-IP.Alternatively,anantibodyagainsttheco-precipitatedproteincanbeusedtoco-IPthesamecomplex.

Determine if the interaction is direct or indirectIstheinteractionmediatedthroughathird-partyproteinthatcontactsbothtargetandco-precipitatedprotein?Immunologicalandothermoresophisticatedmethodssuchasmassspectrometrymaybenecessarytoanswerthisquestion.

Determine that the interaction takes place in the cell and not as a consequence of cell lysis Suggestedapproacheshereinvolveco-localizationstudiesandsite-specificmutagenesisgivingrisetomutantsthatperturbthebindingprocess.

ReferencesAdams,P.D.,et al.(2002).Identificationofassociatedproteinsbyco-immunoprecipitation,In Protein-Protein Interactions – A Molecular Cloning Manual.Golemis,E.,Ed.,ColdSpringHarborLaboratoryPress,pp59-74.

Liebler,D.C.(2002).Identifyingprotein-proteininteractionsandproteincomplexes.In Introduction to Proteomics, Tools for the New Biology,HumanaPress,pp.151-165.(Product#20061)

Phizicky,E.M.andFields,S.(1995).Protein-proteininteractions:methodsfordetectionandanalysis.Microbiological Reviews(Mar.),pp.94-123.

IP/Co-IP


IP and Co-IP Kits

TheThermoScientificPierceClassicandCrosslinkIPKitsareidealwhenusingantibodiesthatbindtoProteinAorProteinG.TheDirectandCrosslinkIPKitsarehighlyeffectiveforeliminatingco-elutionofIgGheavyandlightchainwiththeantigen,whichinterfereswithdownstreamapplicationssuchasmassspectrometryanalysisorproteinsequencing.

Traditionalco-IPmethodsresultindetectionoftheantibodywiththetargetproteins.Becausetheantibodyheavyandlightchainsmayco-migratewithoneoftherelevantbands,importantresultscanbemasked.ThePierceCo-IPKitcircumventsissueswithco-migrationofantibodychainswithtargetproteinsbyretain-ingtheantibodyontheresin.Thenewkitisoptimizedforusingsmalleramountsofsampleandoffersasinglelysis/washbuffereliminatingtheneedforaseparatelysisreagent.

All four IP Kits Highlights:•Requireminimalantibody(2-10μg)•Arehighlyeffectiveandefficientincapturingantigens•UseoptimizedprotocolsandbuffersforefficientIPsand

antigenelution•Usecommonlysis/binding/washbuffer•Includespincolumnsandcollectiontubesthatshortenthe

protocolbyminimizinghandlingandmixing•Useanewelutionbufferthatprovidesmilderandlessdenaturing

recoveryofantibody:antigencomplexes•Arecompatiblewithspecializeddownstreamapplications;e.g.,

massspectrometry,enzymeassaysandantibodyproduction•Areabletoscaleupasneededusingourflexibleprotocol

Comparison of immunoprecipitation methods. Thefollowingtablecomparesthekeyfeaturesoftraditional“do-it-yourself”immunoprecipitationtechniquestotheThermoScientificPierceIPKits.Considerationofthesefeaturescanhelptodeterminewhichmethodismostappropriatefortheavailablereagentsanddownstreamapplication.

Feature

Traditional IP method

Pierce Classic IP Kit

Pierce Crosslink IP Kit

Pierce Direct IP Kit

Pierce Co-IP Kit

Useshighbindingcapacityresin Variable1 Yes(ProteinA/GPlus)

Yes(ProteinA/GPlus)

Yes(AminoLinkPlus)

Yes(AminoLinkPlus)

Crosslinkermediatedimmobilization No No Yes(DSS)

No No

Requirespurifiedantibodyinamine-freeandprotein-freestoragesolution

No No No Yes Yes

Antibodyiscovalentlyattachedtoagaroseresin No No Yes Yes Yes

Antibodyisoriented Yes Yes Yes No No

Antibodyeluteswithantigen Yes Yes No No No

Antigenrecoverymethod Boilingw/SDS(LowpH)

LowpHelution(Boilingw/SDS)

LowpHelution

LowpHelution

LowpHelution

Relativeantigenrecovery2 Variable Highest Medium High High

Immobilizedantibodycanbereused No No Possible3 Possible3 Possible3

Co-purificationofinteractingproteins Possible Possible Possible Optimal

1 Commercially available resins vary in binding capacity and performance for IP assays.2 Antigen yield depends on the activity of the antibody, specific binding conditions and the immobilization method used.3 It is possible to reuse the prepared antibody affinity resin if the antibody remains functional following low pH elution.


Comparison of three different Thermo Scientific Pierce IP Kits. Immunoprecipitationswereperformedaccordingtotheproductinstructionsusing10μgofaffinity-purifiedgoatanti-GFPantibodyandthePierceDirect,ClassicandCrosslinkIPKits.ThecelllysatewaspreparedusingIPLysis/WashBufferandpre-clearedusingthePierceControlAgaroseResinsuppliedinthekits.Theimmunecomplexwasformedbyincubatingtheantibody,resinandlysateovernight.TheresinwaswashedwithIPLysis/WashBuffer,1XConditioningBufferandelutedwithElutionBuffer.Foranalysis,4-20%Tris-glycinegelswereloadedwith20%oftheelutedsample,5%ofthecelllysateload(Lysate)and10%oftheantibodyload(IgG)andstainedwithThermoScientificImperialProteinStain(Product#24615).Fortheresincon-trols,theimmunoprecipitationwasperformedwithoutaddingtheantibody.

Co-immunoprecipitation of interacting proteins using the Thermo Scientific Pierce Co-IP Kit. EpidermalGrowthFactor(EGF)wasco-immunoprecipitatedfrom2,000μgofHeLalysatewith40and20μganti-EpidermalGrowthFactorReceptor(EGFR)mousemonoclonalIgG1+IgG2a(ThermoScientific)usingthePierceCo-IPKit.Co-immunoprecipitationswereperformedaccordingtoproductinstructions.TwoidenticalWesternblotswereprobedwithanti-EGFRsheeppolyclonalIgG(Millipore)oranti-EGFrabbitpolyclonalIgG(SantaCruz).ThePierceCo-IPKitco-immunoprecipitatedEGFwiththeEGF-R.

The Classic IP Kit:Product#26146• High binding-capacity recombinant Protein

A/G Plus Agarose Resin results in higher antigen yields

• Recombinant Protein A/G Plus Resin offers compatibility with a wider range of mamma-lian IgG species for IP reactions (e.g., mouse, rabbit, human and goat IgG subclasses)

The Crosslink IP Kit:Product#26147• Able to purify target protein without con-

tamination by the antibody in the eluate

• Improved crosslinking protocol optimized for maximum antibody functionality

• Bound immunoglobulins oriented for optimal antigen-binding sites are more accessible

• High-capacity Protein A/G Plus Resin allows for better purifi-cation and immobilization of antibodies

• Offers compatibility with a wider range of mammalian IgG species

The Direct IP Kit: Product#26148• Immobilize any antibodies independent

of isotype or species

• Improved antibody coupling protocol

• Purify target protein without antibody contamination

• Activated resin for directly coupling antibodies to the support resin

• Eliminate antibody contamination in the eluate

The Co-IP Kit: Product#26149• Minimal antibody requirements for

co-IP reactions

• Shorter antibody coupling protocol (<2 hours)

• Compatible with any antibody species and subclass

• Requires a purified antibody in a solution free of amines and stabilizing proteins

• Optimized protocols and buffers for efficient co-IP and antigen elution

• Allows for selective purification of target protein

• Includes spin columns and collection tubes that shorten the protocol by minimizing handling and mixing

• Compatible with specialized downstream applications, e.g., mass spectrometry

• Allows for scale up

Bead

Bead

Bead

Bead

Protein A/G PlusAntibodies Alkylamine linkage DSS Crosslinking

GFP (27 kDa)

IgG Heavy Chain(55kDa)

Thermo ScientificPierce IP Kits

Direct

Classic

Lysa

teIgG MW

Direct

Classic

Crosslin

k

Crosslin

k

IgG Light Chain(25kDa)

Resin Controls

EGF-R (170kDa)

µg anti-EGFR

EGF (6kDa)

40 20

IP/Co-IP


Thermo Scientific Pierce Classic IP KitA convenient kit for a new spin on traditional immunoprecipitation.

ThePierceClassicIPKitprovidesallthenecessaryreagents,spincupsandcollectiontubestoperformsuccessfulimmunopre-cipitation(IP)experimentswithease.Thekituseshigh-capacityProteinA/GagaroseaffinityresinforefficientbindingofmostspeciesandsubclassesofIPantibodies.TheincludedIgGelu-tionbufferprovidesmilderandlessdenaturingrecoveryofantibody:antigencomplexesthanthetraditionalmethodofboilinginreducingsamplebufferforSDS-PAGE,facilitatingagreatervarietyofmethodsforsubsequentanalysis.Themicrocentrifugespincolumnformathelpstoensureeffectivewashingandsep-arationofsamplesfromthebeadedagaroseaffinityresin.

LiketraditionalIPmethods,thePierceClassicIPKitprocedureinvolvesformationofantibody:antigencomplexesinasamplesolutionandthencaptureofthatcomplextoanIgG-bindingproteinthatiscovalentlyboundtobeadedagaroseresin(ProteinA/GAgarose).Afterwashingtoremovenonbound(presumablyundesired)componentsofthesample,theantigenandantibodyarerecoveredfromthebeadedresinwithelutionbuffersuppliedinthekit.Theentireprocedureisperformedinamicrocentrifugespincolumn,allowingsolutionstobefullyseparatedfromtheagaroseresinuponbriefcentrifugation.


Description

Pkg. Size

U.S. Price

26146 Pierce Classic IP Kit Sufficient reagents to perform 50 reactions. Includes:PierceProteinA/GPlusAgarose

IPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBuffer5XLaneMarkerSampleBuffer,Non-reducingPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin

Kit0.55mL2x50mL5mL25mL50mL5mL

50each2mL,100each1.5mL,50each2mL

$259

Thermo Scientific Pierce Crosslink IP KitPurify target protein complexes without antibody interference!

TheCrosslinkIPKitextendsthefunctionalityoftraditionalimmuno-precipitation(IP)methodsbyaddingcrosslinkingtechnologyandmicrocentrifugespincolumnsamplehandlingtotheprocedure.Theprimarybenefitsresultingfromthesefeaturesaretheabilitytopurifytargetproteinwithoutcontaminationbytheantibodyandtheabilitytomoreeffectivelywashandseparatesamplesfromthebeadedagaroseresin.

ThePierceCrosslinkIPKitmethodinvolvescapturingtheIPanti-bodytoProteinA/GAgaroseresinandcovalentlyimmobilizingittothesupportbycrosslinkingwithdisuccinmidylsuberate(DSS).Theantibodyresinisthenincubatedwiththesamplethatcontainstheproteinantigenofinterest,allowingtheantibody:antigencom-plextoform.Afterwashingtoremovenonbound(presumablyundesired)componentsofthesample,theantigenisrecoveredbydissociationfromtheantibodywithelutionbuffersuppliedinthekit.Theentireprocedureisperformedinamicrocentrifugespincup,allowingsolutionstobefullyseparatedfromtheagaroseresinuponbriefcentrifugation.Onlyantigeniselutedbytheprocedure,enablingittobeidentifiedandfurtheranalyzedwithoutinterfer-encefromantibodyfragments.Furthermore,theantibodyresinoftencanbereusedforadditionalroundsofimmunoprecipitation.


Description

Pkg. Size

U.S. Price

26147 Pierce Crosslink IP Kit Sufficient reagents to perform 50 reactions. Includes:PierceProteinA/GPlusAgarose

20XCouplingBufferDSSCrosslinker,No-Weigh™FormatIPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBufferLaneMarkerSampleBuffer,Non-reducing,(5X)PierceSpinColumns-ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin

Kit0.55mL25mL8x2mg2x50mL5mL25mL50mL5mL

50each2mL,100each1.5mL,50each2mL

$305


Thermo Scientific Pierce Direct IP KitImmunoprecipitate using any antibody species or subclass! Eliminate antibody band contamination of IP products.

ThePierceDirectIPKitrepresentsasignificantadvancementinimmunoprecipitation(IP)technologybyreplacingtheuseofimmobilizedProteinAorProteinGwithamethodfordirectcovalentattachmentofantibodiestothebeadedagaroseresin.

Theprimarybenefitsresultingfromthismethodaretheopportunitytouseanyspeciesorsubclassofpurifiedantibody(notjusttypesthatbindtoProteinAorG)andtheabilitytopurifytargetproteinwithoutcontaminationbytheantibody.Themethodalsomakesitpossibletoimmunoprecipitateantigensfromserumsampleswithoutco-purifyingnon-targetimmunoglobulins.Finally,thekitusesmicrocentrifugespincupstoeffectivelywashandseparatesamplesfromthebeadedagaroseresin.


Description

Pkg. Size

U.S. Price

26148 Pierce Direct IP Kit Sufficient reagents to perform 50 reactions. Includes:AminoLinkPlusCouplingResin

20XCouplingBufferQuenchingBufferWashSolution5MSodiumCyanoborohydrideSolutionIPLysis/WashBuffer100XConditioningBuffer20XTris-BufferedSalineElutionBuffer5XLaneMarkerSampleBufferPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubesPierceControlAgaroseResin

Kit2mL25mL50mL50mL0.5mL2x50mL5mL25mL50mL5mL50each2mL,100each1.5mL,50each2mL

$259

Thermo Scientific Pierce Co-Immunoprecipitation KitPerform co-immunoprecipitation experiments without antibody interference.

ThePierceCo-Immunoprecipitation(Co-IP)Kitenablesisolationofnativeproteincomplexesfromalysateorothercomplexmixturebydirectlyimmobilizingpurifiedantibodiesontoanagarosesupport.

Co-IPisacommonapproachtostudyprotein:proteininterac-tionsthatusesanantibodytoimmunoprecipitatetheantigen(baitprotein)andco-immunoprecipitateanyinteractingproteins(preyproteins).Traditionalco-IPmethodsthatuseProteinAorGresultinco-elutionoftheantibodyheavyandlightchainsthatmayco-migratewithrelevantbands,maskingimportantresults.ThePierceCo-IPKitresolvesthisissuebycovalentlycouplinganti-bodiesontoanamine-reactiveresin.Thekitincludesoptimizedbuffersforproteinbindingandrecovery,reagentstoperformcon-trolexperimentsandefficientspincolumnsandcollectiontubes,whichshortentheprotocolandminimizehandlingandmixing.


Description

Pkg. Size

U.S. Price

26149 Pierce Co-Immunoprecipitation Kit* Sufficient reagents to perform 50 reactions. Includes:AminoLinkPlusCouplingResin

20XCouplingBuffer5MSodiumCyanoborohydrideSolutionQuenchingBufferWashSolutionIPLysis/WashBuffer100XConditioningBufferElutionBuffer5XLaneMarkerSampleBuffer,Non-reducingPierceControlAgaroseResin20XModifiedDulbecco’sPBSBufferPierceSpinColumns–ScrewCapMicrocentrifugeCollectionTubesMicrocentrifugeSampleTubes

Kit2mL25mL0.5mL50mL60mL2x50mL5mL50mL5mL2mL25mL50each100each50each

$308

* The Thermo Scientific Pierce Co-IP Kit (Product # 23600) and the Pierce Mammalian Co-IP Kit (Product # 23605) were discontinued on December 31, 2009. The next-generation Pierce Co-IP Kit (Product # 26149) will replace these old kits. The new co-IP kit is optimized for using smaller amounts of sample and offers a common lysis/wash buffer eliminating the need for a separate lysis reagent (Thermo Scientific M-PER Mammalian Protein Extraction Reagent, Product # 78503) as was offered with the Mammalian Co-IP Kit.

IP/Co-IP


Thermo Scientific Pierce Coated Plate Immunoprecipitation KitsPre-coated 96-well plates are easier to use and faster than traditional microcentrifuge tube methods.

ThermoScientificPierceCoatedPlateIPKitsenablerapidimmunoprecipitationofmultiplesampleswithouttheusualtediumofpipetting,centrifugingandseparatingbeadedaffinityresininindividualmicrocentrifugetubes.Immunoprecipitationisaccomplishedusingcoated96-wellmicroplatesratherthanbeadedagaroseresin.Theplateformatallowsfastprocessingofmultiplesamples.SelectfromProteinA/G-,ProteinG-orstreptavidin-coatedplates.

Highlights:•Ready-to-use,high-qualitycoatedplatesprovidehighcapacity andconsistency•Plateformatbestsuitedforsimultaneouslyprocessingmultiple samplesandtheircontrolconditions•Faster,easierandmorethoroughwashingthanwithtraditional tube/resinIPmethods•UsesfamiliarandconvenientELISAtools(multichannelpipettors andplatewashing);notediousseparationofsupernatantfrom pelletedresinbeads,andnotubestoopen,closeandcentrifuge• Coatedplatesare96-wellstripplates,convenientforexperi-

mentsrequiringonlyapartialplate•Easy-to-followinstructions,includingdetailedexplanationof appropriatecontrols•Threekitsavailable,suitableformostcommonantibodytypes (mouse,rabbit,humanandgoatIgGsubclasses)orany biotinylatedantibodyor“bait”protein

3 4 521

Thermo Scientific Pierce Protein A/G Coated Plate IP Kit (Product # 45350) immuno precipitation of CD71 (transferring receptor) from human serum using and a goat anti-CD71 polyclonal antibody.Elutedproductsfortheexperimentalandcontrolsamplesweremixedwithnonreducingsampleloadingbuffer,separatedbySDS-PAGE,transferredtonitrocellulosemembraneanddetectedbyWesternblottingwiththeIPantibody,Goat-anti-mouse-HRPconjugatedsecondaryantibody(Product#31432)andThermoScientificSuperSignalWestDuraChemiluminescentSubstrate(Product#34076).Lane 1:Experiment(immunoprecipitationproduct)Lane 2:Antibody-onlycontrol(nosample)Lane 3:Humanserumsamplecontrol(noantibody)Lane 4:Platecontrol(noantibodyorhumansample)Lane 5:Puretargetprotein(CD71)forsizereference

Choosing between Protein A/G and Streptavidin Coated Plate KitsStreptavidinisaproteinthatbindsspecificallyandstronglytobiotin;therefore,theStreptavidinCoatedPlateIPKit(Product#45360)isappropriateforimmunoprecipitationwhenusingabiotin-labeled(biotinylated)antibody.Thiskitcanbeusedtoaffinity-purifyabindingpartnertoanyantibodyspeciesorsubclassoranyotherproteinormoleculethatisbiotinylated.Becausethestreptavidin-biotinaffinityinteractionissostrong,theelutionstepgenerallywilldissociateonlytheantigen(bindingpartner),notthebiotinylatedantibodyor“bait”protein.

ProteinAandProteinGaredifferentproteinsthatbindtoimmuno-globulins(primarilyIgG).Typically,ProteinAispreferredforusewithrabbitpolyclonalantibodies,whileProteinGispreferredforusewithmouseantibodies(especiallymonoclonalsoftheIgG1subclass).ProteinA/GisarecombinantofProteinAandProteinGthathastheadditivebindingpropertiesofbothproteins.

ReferenceDesai,S.andHermanson,G.(1997).Previews1(3),2-7.


Description

Pkg. Size

U.S. Price

45350 Pierce Protein A/G Coated Plate IP KitAntibody binding capacity/well: 2.5µg. Sufficient capacity for downstream analysis of IP or co-IP proteins in-gel or by Western blot.Includes:ProteinA/GCoated12x8-wellstripplates Phosphatebufferedsaline Surfact-Amps®X-100(10%TritonX-100) Elutionbuffer Neutralizationbuffer Uncoated96-wellstripplates(white), (forsamplecollectionandneutralization) Platesealers

Kit

2plates2packs6x10mL50mL7mL2ea.

18sheets

$295

45360 Pierce Streptavidin Coated Plate IP KitAntibody binding capacity/well: 5µg. Sufficient capacity for downstream analysis of IP or co-IP proteins in-gel or by Western blot.Includes: HighBindingCapacityStreptavidin CoatedPlates Biotinblockingbuffer Phosphatebufferedsaline Surfact-AmpsX-100(10%TritonX-100) Elutionbuffer Neutralizationbuffer Uncoated96-wellstripplates(white), (forsamplecollectionandneutralization) Platesealers

Kit

2plates30mL2packs6x10mL50mL7mL2ea.

18sheets

$298


Thermo Scientific Pierce HA- or c-Myc Tag IP/Co-Immunoprecipitation KitsNeed to perform IP or co-IP reactions with your HA- or c-Myc-tagged protein? Open box ... Read instructions ... Start performing an IP or co-IP. High-specificity immobilized antibodies make it easy.

Notagsaremorepopularformammaliansystemproteinexpres-sionthanHAorc-Myc.Althoughthesetagsareextremelypopular,akitthatallowsyoutoconvenientlyperformimmunoprecipitation(IP)orco-immunoprecipitation(co-IP)reactionsusingthesetagshasnotbeenavailable.ThePierceIP/Co-IPKitsincludeallneces-saryreagents,buffersandhardwarethatallowefficientpurifica-tionofataggedtargetprotein(i.e.,IP)orconfirmationofpotentialinteractionsindicatedbyyeasttwo-hybridresults(i.e.,co-IP).

Thesefourkits,whicharespecificallyforHA-andc-Myc-taggedproteins,allowyoutoeasilyperformanIPorco-IPexperimentwithminimaloptimization.High-affinity,high-specificityantibod-iesimmobilizedontoanagarosematrixareattheheartofthesenewkits.Inaddition,thekitscontainafullcomplementofbuffers,eluents,apositivecontrolandnecessaryhardwaretoefficientlyperformtheintendedapplication.

Highlights:IP and co-IP for HA- or c-Myc-tagged proteins directly out of the box.• DemonstratedutilityintheIPandco-IPapplicationbenefitsthe

noviceandexpert.Ourkitsincludeallessentialcomponentstoperformtheassays.There’snoneedtoformulateorvalidaterawmaterials.

Immobilized high-affinity antibodies with excellent specificity for the HA or c-Myc tag.• Theimmobilizedanti-HAandanti-c-Mycmonoclonalsprecipitate

theappropriatelytaggedproteinspecificallyandinhighyield,resultingincleanWesternblotdetection.

• Excellentresultswithaslittleas2.5μgofanti-HAantibodyand1μgofanti-c-MycantibodyintheIPmodewiththerespectivepositivecontrollysate.

• Limitspossibilityofnonspecificbindingtootherproteinsinthelysate.

• Eliminatescontaminationfromantibodyorantibodyfragmentsafterelutionoftheprecipitatedproteinortheco-IPcomplex.Thisbenefitisespeciallyimportantwheninterpretingproteininteractionresults.

Simple, flexible and easy-to-follow protocols.• Completekitformatoffersoptimumconvenienceinboththe

IPandco-IPmodes.• EliminateProteinAorProteinG,reducingnonspecificbinding

andshorteningtheIPprocedure.• Systemdemonstratesexcellentflexibilitywithrespecttothe

amountofantibodyoramountoflysateused,enablingisolationoflow-expressionHA-/c-Myc-taggedtargets.

• TheuseofSpinColumnsacceleratetheIP/co-IPprocess.Spin Columns.•SpinColumnsareveryconvenientforsmallsamplehandling.•Allowmoreefficientwashing.•Eliminateresinlosses

HA- or c-Myc Tag IP/Co-IP Kit Descriptions Eachkitlistedatrightconsistsoftwocomponents:anApplicationSetandaPositiveControlLysate.TheApplicationSetcontainstheimmobilizedsupportappropriateforthekitandalloftherequiredbuffers,eluentsandhardware.ThesecondcomponentisabacteriallysatecontaininganoverexpressedGSTwitheitherHAorc-MycastheC-terminaltag.ThemammalianversionofeachkitcontainsThermoScientificM-PERProteinExtractionReagentforusewithmammaliancell-basedIPorco-IPapplications.TheApplicationSetsandPositiveControlLysatescanalsobeorderedseparately.

1. Lyse cells expressing HA- or c-Myc-tagged protein.

2. Combine cell lysate and Immobilized Anti-HA or Anti-c-Myc.

3. Incubate at 4˚C for 1 hour to overnight with end-over-end mixing.

4. Pulse centrifuge for 10 seconds.

5. Wash three times with TBS-T. Pulse centrifuge for 10 seconds after each wash.

6. Elute HA- or c-Myc-tagged protein using Elution Buffer or Non-Reducing Sample Buffer.

CollectionTube

Bottom PlugRemoved

Thermo Scientific Pierce HA- or c-Myc IP/Co-IP Kit Protocol summary.

GST-c-MycGST-HA

IP

MW

Mar

ker

HA

Lys

ate

Elut

ion

#1

Elut

ion

#2

c-M

yc L

ysat

e

Elut

ion

#1

Elut

ion

#2

IP

Effectiveness of elution options in the IP of GST-HA and GST-c-Myc from bacterial lysates.IPresultsachievedwiththeThermoScientificPierceHAandc-MycIP/Co-IPKitsusingtheappropriatepositivecontrollysateprovidedandsuggestedelutionoptionsforGST-HAandGST-cMyc,respectively.Theelutioncomponentsaresuppliedwitheachkit.Elutionperformedwith#1.ElutionBufferor#2.2Xnonreducingsamplebuffer.

IP/Co-IP



Description

Pkg. Size

U.S. Price

23610 HA-Tag IP/Co-IP Kit Sufficient material to conduct 25 IP/co-IP assays using proteins expressed with an HA tag. Kit is supplied complete with an HA-tagged positive control lysate.Includes:ImmobilizedAnti-HA(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent HA-TaggedPositiveControl

Kit

150μL1pack50mL5mL

25mL

500μL

$399

23615 Mammalian HA-Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with an HA tag. Kit is supplied complete with a mammalian cell lysis buffer and an HA-tagged positive control lysate.Includes:ImmobilizedAnti-HA(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent HA-TaggedPositiveControl

Kit

150μL1pack50mL5mL

25mL

500μL

$423

23620 c-Myc-Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with a c-Myc tag. Kit is supplied complete with a c-Myc-tagged positive control lysate.Includes:ImmobilizedAnti-c-Myc(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps c-Myc-TaggedPositiveControl

Kit

250μL1pack50mL5mL

500μL

$399

23625 Mammalian c-Myc Tag IP/Co-IP KitSufficient material to conduct 25 IP/co-IP assays using proteins expressed with a c-Myc tag. Kit is supplied complete with a mammalian cell lysis buffer and a c-Myc-tagged positive control lysate.Includes:ImmobilizedAnti-c-Myc(agaroseresin) BupHTrisBufferedSalinePack ElutionBuffer,pH2.8 LaneMarkerNon-ReducingSampleBuffer(5X) SpinColumnsAccessoryPack, 27columnswithpre-insertedfrit andtopandbottomcaps CollectionTubesandCapsAccessoryPack, 100graduated2mLtubesandplugcaps M-PERMammalianProteinExtraction Reagent c-Myc-TaggedPositiveControl

Kit

250μL1pack50mL5mL

25mL

500μL

$423

Protein Interactions Technical Handbook

Our72-pageProteinInteractionTechnicalHandbookprovidesprotocolsandtechnicalandproductinformationtohelpmaximizeresultsforProteinInteractionstudies.Thehandbookprovidesbackground,helpfulhintsandtroubleshootingadviceforimmunoprecipitationandco-immunoprecipitationassays,pull-downassays,Far-Westernblottingandcrosslinking.Thehandbookalsofeaturesanexpandedsectiononmethodtostudyprotein:nucleicacid

interactions,includingChIP,EMSAandRNAEMSA.Thehand-bookisanessentialresourceforanylaboratorystudyingProteinInteractions.(#1601618)


Protein Enrichment

Protein Enrichment

Protein enrichment encompasses numerous techniques to isolate subclasses of cellular proteins based on their unique biochemical activity, post-translational modification (PTM) or spatial localization in a cell. Protein enrichment is essential for studying low abundant proteins and for reducing the complexity of samples for proteomic analysis. Enrichmentofspecificproteinsorproteincomplexescanmosteasilybeaccomplishedusingimmunoaffinitytechniquessuchasimmunoprecipitationandco-immunoprecipitation.Althoughtheseantibody-basedtech-niquesarewidelyused,elutionofimmunoprecipitatedproteinscansometimesresultinlowproteinrecoveryorantibodycontami-nationinsamples.

Globalproteinenrichmentstrategiesinvolvetheselectiveisola-tionofdistinctproteinsubclasseswhichshareacommonpost-translationalmodificationorcellularlocalization.Post-translationalmodificationssuchasphosphorylationandglycosylationcanbeenrichedusingaffinityligandssuchasion-metalaffinitychroma-tography(IMAC)orimmobilizedlectins,respectively.Inaddition,PTM-specificantibodieshavebeenbeused.OthertechniquesusemetabolicorenzymaticincorporationofmodifiedaminoacidsorPTMstointroduceuniqueproteinchemistrywhichcanbeusedforenrichment.Finally,proteinscanalsobeenrichedusingvariousenzymeclassspecificcompoundsorcell-impermeablelabelingreagentswhichselectivelylabelcellsurfaceproteins.

Phosphoprotein Enrichment Kits

Process cell and tissue samples in less time and with greater purity.

Phosphorylationisoneofthemostfrequentlyoccurringpost-translationalmodificationsinproteins.Itisestimatedthatasmanyas30%ofallcellularproteinsaretransientlyphosphorylatedonserine,threonineandtyrosineresidues.

Reversibleproteinphosphorylationregulatesnearlyallintra-cellularbiologicalevents,includingsignaltransduction,protein-proteininteractions,proteinstability,proteinlocalization,apoptosisandcell-cyclecontrol.Deregulationofproteinphosphorylationisahallmarkofnumeroushumandiseases,includingcancerandmetabolicandimmunedisorders.

Detectingchangesinproteinphosphorylationcanbeadifficulttaskbecauseofthetransientlabilestateofthephosphategroup.Furthermore,lowphosphoproteinabundanceandpoorlydevelopedphospho-specificantibodiescontributetodifficultiesinphosphoproteindetection.Recentadvancesinmassspectrometrytechnologyincombinationwithphosphoproteinenrichmentusingimmobilizedmetalaffinitychromatography(IMAC)haveresultedingreaterresolutionofthephosphoproteome.

ThenewThermoScientificPiercePhosphoproteinEnrichmentKitefficientlyenrichesphosphorylatedproteinsderivedfrommammaliancellsandtissues.Theproprietarymetalandbuffercompositionproducessuperioryieldswithnegligiblenonspecificbinding.

Highlights:• Specific–lowcontaminationfromnonspecificproteins• Fast–easy-to-usespinformatenrichesofphosphorylated

proteinsinlessthan2hours• Superior yield–highyieldfromcomplexbiologicalsamples,cell

culturelysateandmousetissueextract• Convenient format–completekitincludespre-dispensedspin

columns,buffers,reagentsandThermoScientificPierceProteinConcentrators

• Compatible–workswithdownstreamapplications,includingmassspectrometry,Westernblottingand2D-PAGE

Phospho-specificantibodiesrecognizingkeyregulatoryproteinsinvolvedingrowthfactorsignalingwereusedtomonitorbindingspecificityofourPhosphoproteinEnrichmentKit(Figure1).SpecificityofthekitisfurtherdemonstratedbytheabsenceofCytochromeC(pI9.6)andp15Ink4b(pI5.5),twoproteinsnotpredictedtobephosphorylated,intheelutionfractionandtheiremergenceintheflow-throughandwashfractions(Figure1).Furthermore,dephosphorylationofHeLacellextractin vitroresultedindiminishedbindingofPTEN,MAPKandGSK3βtothePiercePhosphoproteinEnrichmentColumnasevidencedbytheirabsenceintheelutionfraction.Conversely,allthreeproteinswerepresentintheelutionfractionfromnon-treatedHeLaextract(Figure2).OurPhosphoproteinEnrichmentKitprovidedsuperiorandefficientphosphoproteinenrichmentyieldswhencomparedtocompetitors’products(Table1).Italsoeffectivelyenrichedphos-phoproteinsfromhomogenizedmouselivertissue(Figure3).


Table 1. The Thermo Scientific Pierce Phosphoprotein Enrichment Kit provides higher phosphoprotein yields in less time than competitors’ kits.

Kit Yield (%) Enrichment Time (Hours)

ThermoScientificPiercePhosphoproteinEnrichmentKit

15 1.5

SupplierQKit 4.4 4.5

SupplierIKit 2.6* 3.5

SupplierCKit 8 3

SupplierEKit Toodilutetodetermine 5

*Based on maximum 1mg load per manufacturer’s protocol.

Phospho-MAPK T202/Y204

Phospho-PTEN S380

Phospho-GSK3β S9


Phospho-Akt S473

Phospho-Src Y527

Cytochrome C

p15Ink4b

NIH

3T3

+ P

DG

F

H

eLa

+ EG

F

FT W E L

Figure 1. Highly pure phosphoprotein enrichment from complex biological samples. Serum-starvedHeLaandNIH3T3cellswerestimulatedwithEGFandPDGF,respectively.Celllysate(2mg)wasusedforenrichment.Concentratedflow-through,washandelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Westernblotanalysiswasperformedusingantibodiesthatdetectsite-specificphosphory-lationevents.CytochromeC(pI9.6)andp15Ink4b(pI5.5)servedasnegativecontrolsfornonspecificbindingofnon-phosphorylatedproteins.FT =flow-throughfraction,W =pooledwashfractions,E =pooledelutionfractionsandL =non-enrichedtotalcelllysate.

Phospho-PTEN S380


Phospho-GSK3β S9

Total Cell Lysate Elution25 µg 10 µg 10 µg

λ Pase - + - + - +

Figure 2. Highly specific phosphoprotein purification from lambda phosphatase-treated cells.Non-treatedandlambdadephosphorylatedHeLacellextract(2mg)wasloadedontoseparateThermoScientificPiercePhosphoproteinEnrichmentColumns.ConcentratedelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Todetermineenrichment,10μgand25μgofnon-treatedorlambdaphosphatase-treatedtotalcellextract(non-enriched)wasloadedontoeachgel.Westernblotanalysiswasperformedusingphospho-specificantibodiesrecognizingkeyproteinsintheRas-MAPKandPI3K-Aktsignalingcascades.

L10 FT W E L25

Phospho-PTEN S380

Phospho-Rb S795

Cytochrome C

Figure 3. Efficient enrichment of phosphoproteins from mouse liver extract. Homogenizedmouseliverextract(~2mg)wasloadedontoaThermoScientificPiercePhosphoproteinEnrichmentColumn.Concentratedflow-through,washandelutionfractionswereresolvedbySDS-PAGE.Gellaneswerenormalizedbyproteinconcentration(10μg/lane).Westernblotanalysiswasperformedusingantibodiesthatdetectsite-specificphosphorylationevents.CytochromeC(pI9.6)servedasanegativecontrolfornonspecificbinding.L10 =non-enrichedtotalcellextract(10μg),FT =flow-throughfraction,W =washfraction,E =elutionfractionandL25 =non-enrichedtotalcellextract(25μg).


Description

Pkg. Size

U.S. Price

90003

Pierce Phosphoprotein Enrichment KitIncludes:PhosphoproteinEnrichmentColumn

ResinBed(1mL)Lysis/Binding/WashBufferElutionBufferCHAPSWhiteColumnCapsPierceProteinConcentrator7mL/9KMWCO

Kit10ea.325mL60mL1g10caps10Devices

$464


Phosphoprotein Pull-Down with SH2 Domains

SH2 Domain Phosphotyrosine Capture Kits

SH2domainsprovideanimprovedapproachtoselectivelymonitorreceptortyrosinekinasesignaling,aswellasthebindingofdown-streameffectorproteins.TheinteractionsofSH2domain-contain-ingproteinsrepresentacriticalinterfacebetweenextracellularstimulationofamembranereceptorandtransmissionofthatsignaltointracellularproteins.

OurkitsincludeoptimallevelsofpurifiedGST-fusedSH2domainsofvarioussignalingproteinsintegraltocellbiology.Eachvalidatedkitincludesoptimizedbuffersandcolumnstoperformproteinpull-downs.UsingSH2domainseliminatesthebackgroundassociatedwithlow-specificityantibodiesandenablesanalysisofreceptortargetstowhichantibodiesarenotavailable.Usingquality-testedpurifiedGST-SH2domainsalsoensuresuniformresultswithoutvariability.

SH2 domains specifically bind phospho-tyrosine residues. EachSH2domainisspecificforitsnaturaltarget(Figure4).In vivo,thephosphataseShp2isrecruitedtotyrosine1009oftheplatelet-derivedgrowthfactorreceptor(PDGFR)onlywhenitisphosphorylated.Also,PLCgisrecruitedtotyrosine1021onPDGFRinresponsetogrowthfactorsignaling.Inquiescentcellsbothtyrosine1009and1021arenotphosphorylatedand,therefore,bothdomainsareunabletobind.

PDGF

Antibody

GST-Shp2 GST-PLCγ

L PD L PD

- +

L PD L PD

- +

pPDGFR Y1009 pPDGFR Y1021

Figure 4. Site-specific interaction of GST-Shp2 and GST-PLCg SH2 on the PDGF-receptor.NIH3T3cellswererenderedquiescentbyserumwithdrawalfor48hoursfollowedbystimulationwithPDGF(50ng/mL,CellSignalingTechnology[CST])for15minutesornostimulation.Eachcelllysate(500μg)wasincubatedovernightat4°Cwitheither100μgGST-PLCg1orGST-Shp2.ProteincomplexeswerecapturedonimmobilizedglutathionebeadsandresolvedbySDS-PAGE.Westernblotanalysiswasperformedusingphospho-specificantibodiesthatdetectknownprotein-proteininteractionsequences(phospho-PDGFRY1009andphospho-PDGFRY1021,CST).Lanes:L = 25μgoftotalcelllysateandPD =SH2domainpull-down.

pY

pY

pY

pY

pY

pY SH2

SH2

Receptor in cell membrane

Extracellular signal

Tyrosine phosphorylation

SH2 domains recognize pY

Other proteins recruited

Translation, Transcription, Cell Proliferation, Differentiation, Apoptosis

Cellular pathway activation

SH2 domains provided a more efficient method of capturing site-specific phospho-tyrosine events as compared to antibody based co-immunoprecipitation. Co-immunoprecipitationexperimentsaredependentonthequalityandspecificityoftheantibodyused.Antibodiesagainstphosphoproteinsarenotoriouslydifficulttoworkwith,yieldinghighbackgroundandlowtargetproteinrecovery.SH2domainsprovidebettercaptureefficiencyascomparedtomanypan-andsite-specificphospho-antibodiesbecauseanSH2domainismoreselectivebynature.Becauseofthisimprovedspecificity,SH2domainscanbeusedtopull-downphosphotyrosinecontainingproteinswithimprovedresultsovertraditionalantibody-basedmethods.InFigure5,PDGF-stimulatedNIH3T3cellswerelysedandincubatedwitheitheraspecificSH2domainorapan-antibodycorrespondingtotheSH2domaincontainingprotein.BindingofeachspecificSH2domaintothePDGFreceptorwasconfirmedbyWesternblotusingapan-PDGFRantibody.InallcasesweobservedahighercaptureefficiencyusingtheSH2domainapproachascomparedtothetraditionalantibodyco-immunopre-cipitation.Inmanycases,wewerealsoabletodetectdifferentialbindingoftheSH2domaininthepresenceofPDGFstimulationascomparedtotheunstimulatedstate.Thesedifferenceswerenotapparentusingtheco-immunoprecipitationapproach.WefurthertestedtheinteractionspecificityoffourSH2domainsusingphospho-antibodieswhichdetectsite-specifictyrosinephosphorylationeventswhichmediateeachSH2domaininteraction.Wewerelimitedtoonlyfourphospho-antibodiesagainstspecifictyrosinesitesonthePDGFreceptorbecauseofcommercialavailabilityand/orantibodyintegrity.Usingthesesite-specificantibodiesweconfirmedtheselectivityandspecificityofeachSH2domaintestedandtheseinteractionsweremuchstrongerascomparedtothetraditionalantibodyimmunoprecipi-tation.TakentogethertheseresultsclearlydemonstratethattheSH2domainpull-downapproachrepresentsarobustmethodtocaptureandmonitorsitespecifictyrosinephosphorylationeventswhicharecriticalforcellhealth.


Figure 5. SH2 domains provide better enrichment of phosphorylated receptor tyrosine kinase (PDGFR) compared to antibody-based pull-down assays. Panel A. NIH3T3cellswererenderedquiescentbyserumstarvationfor24hourswithDMEMcontaining0.1%FCS.Followingstarvationcellswereeitherstimulated(100ng/mLPDGFfor20minutes)orleftunstimulated.CellswerethenlysedinNP40lysisbuffer(50mmTrispH7.5,150mmNaCl,1%NP40,5%glycerol)containingThermoScientificHaltProteaseandPhosphataseInhibitorCocktail(Product#78440)andproteinconcentra-tionsdeterminedbytheThermoScientificPierce660nmAssay.SH2andco-immunoprecipitationswereperformedasfollows:SH2domainpull-downsconsistedof100μgofSH2domain(Abl,Crk,Grb2,Lyn,PI3K,Plc-gamma,RasGap,Shc,Shp2,Src)addedto250μgofstimulatedorunstimulatedlysate.Co-immunoprecipitationswereperformedbyincubating10μLofpanantibody(Abl,Crk,Grb2,Lyn,PI3K,Plc-gamma,RasGap,Shc,Shp2,Src)with250μgofstimulatedornon-stimulatedlysate.Bindingwasperformedfor16hoursat4°Conarotatingplatform.Followingbinding100μLofa50%slurryofimmobilizedglutathionewasaddedtotheSH2domainsamplesand100μLofa50%slurryofProteinA/Gwasaddedtotheco-immunoprecipitationsamples.Sampleswere

incubatedfor1hourat4°Conarockingplatformfollowedbycentrifugationat1000xgfor1minutetocollectcomplexesboundtotheresin.Resinbedswerethenwashed3timeswith500μLofThermoScientificM-PERWashBuffer.Toeluteboundproteins25μLof5XDTTloadingdyewasaddedtoeachsampleandboiledfor5minutes.ProteincomplexeswerethenresolvedbySDS-PAGEona4-20%Tris-glycinegel.ProteinsweretransferredontoPVDFmembranefor16hoursat4°C,30V.Membraneswereblockedin7.5%BSAinTBSTandWesternblotsperformedusinga1:2000dilutionofmousemonoclonalanti-PDGFR-betaantibody(CST3175).DetectionwasperformedusingThermoScientificSuperSignalPicoChemilumescentDetectionSystem.Panel B. MembranescontainingtheGrb2,Plcg,PI3K,Src,andRasGappull-downswerestrippedwithThermoScientificRestorePlusBufferandreprobedwithphospho-antibodieswhichdetectsite-specifictyrosinephos-phorylationeventsonthePDGFreceptorwhichmediatetheSH2domaininteraction.TheseantibodiesincludepPDGFRY716,pPDGFRY751,pPDGFRY1009,pPDGFRY579whichcorrespondtoSH2domaindockingsitesforGrb2,PI3K,Plcg,andSrcrespectively.DetectionwasperformedusingSuperSignal®WestPicoChemilumescentDetectionSystem.

PDGF - + - + - + - + - + - + - + - + - + - +

PDGF - + - + - + - + - + - + - + - + - + - +

SH2 IP SH2 IP SH2 IP SH2 IP SH2 IP

SH2 IP SH2 IP SH2 IP SH2 IP SH2 IP

Abl Crk Grb2 Lyn PI3K

Plcγ RasGap Shc Shp2 Src

Panel A.

Panel B.

Western Blot: Pan PDGFR

Western Blot: Pan PDGFR

PDGFR

PDGFR

SH2: Grb2IP: Pan Grb2 WB: pPDGFRY716

SH2: PI3KIP: Pan PI3K WB: pPDGFRY751

SH2: PlcγIP: Pan PlcγWB: pPDGFRY1009

SH2: Src IP: Pan Src WB: pPDGFRY579

PDGF - + - + - + - + - + - + - + - +SH2 IP SH2 IP SH2 IP SH2 IP

pPDGFR


Perform receptor phosphorylation time-course experiments. WithSH2domainpull-downs,itispossibletomonitortime-dependentbindingofSH2domainstotheirtarget.Forexample,PLCg,SrcandShcbindrapidlytothePDGFreceptorinresponsetoPDGFstimulation(Figure6).Innature,phosphorylationisrapidandtran-sient,asevidencedbydiminishedbindingat10-and20-minutepost-stimulation.

MinutesPost PDGF

Stimulation

GST-PLCγ

GST-Src

GST-Shc

0 1 5 10 20

Figure 6. Time-specific binding of various SH2 domains to the PDGF receptor. NIH3T3cellswererenderedquiescentbyserumwithdrawalfor48hours.Post-starvationcellswerestimulatedwithPDGF(50ng/mL,CST)fortheindicatedtimesoruntreated.SH2domainpull-downswereperformedwith100μgofeachSH2domainand250μgofeachcelllysate.ProteincomplexeswereresolvedbySDS-PAGEandanalyzedbyWesternblotusingapanantibodyrecognizingPDGFR(CST).

Resolve specific tyrosine phosphorylation events. SH2domainaffinityprovidesalevelofselectivitynotpreviouslyattainablewithgenericanti-phosphotyrosineantibodies.UsedifferentSH2domainstouncoverthemechanismofhowsignalsaretransducedortoscreenmultipleSH2domainsagainstaparticulartargetatonetime.Forexample,differentnaturallyoccurringSH2domainsefficientlybindtotheepidermalgrowthfactorreceptor(EGFR)inresponsetoEGFstimulation(Figure7).Therewerestronginteractionsofmanyofthedomainsinthepresenceofepidermalgrowthfactor(EGF)andlow-levelbindinginquiescent,serum-starvedA431cells.RasGapbindingisindependentofEGFstimulation,whereasGrb2bindingishighlyupregulated(Figure7).


Description

Pkg. Size

U.S. Price

87700 Grb2 SH2 Domain Phosphotyrosine Capture KitSufficient reagents for six pull-down reactions (100µg/pull-down).Includes:GST-Grb2SH2Domain(37kDa)

GST(negativecontrol,27kDa)ImmobilizedGlutathioneResin(50%resinslurry)SpinColumn(0.8mL)M-PERMammalianProteinExtractionReagentLaneMarkerReducingSampleBuffer(5X)

6pull-downs

600μgat1mg/mL200μgat1mg/mL400μL8each25mL200μL

$408

87701 Src SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87702 Abl SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87703 Crk SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87704 Fyn SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87705 Lck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

L25

*

* * *

* **

EGF

EGF

+ – + – + – + – + – + –

EGF

* = Known Interactions

Antibody: Pan EGFR

** = GST Only: Negative Control

L25+ –

Shp2+ –

Grb2 Src PLCγ Hck Fyn

L25+ – + – + – + – + – + –

Gap Abl Crk GST Nck

Figure 7. High efficiency binding of different naturally occurring SH2 domains to EGFR in response to EGF stimulation. A431cellswererenderedquiescentbyserumwithdrawalfor48hoursfollowedbystimulationwithEGF(100ng/mL)for15minutesorleftuntreated.Eachcelllysate(500μg)wasincu-batedwith100μgofeachGST-SH2domainovernightat4˚C.ProteincomplexeswerecapturedonimmobilizedglutathionebeadsandresolvedbySDS-PAGE.WesternblotanalysiswasperformedusingapanantibodythatrecognizestheEGFreceptor.L25=25μgtotallysateload.

Identify binding interactions with MS. Pull-downsamplesisolatedwithSH2domainsarecompatiblewithMSanalysisforidentifyingproteininteractingpartners.

ImprovedspecificitywithSH2domainsandtheeliminationofantibodycontaminationtypicallyseenintraditionalimmunopre-cipitationexperimentsdeliverpureproteinforMSanalysis.TwodifferentSH2domainswereusedtopulldowninteractingproteinsfromNIH3T3celllysates,whichwerethenanalyzedbyMS.ManyoftheinteractingproteinsidentifiedarespecifictoeitherFgrorPLCg.


Description

Pkg. Size

U.S. Price

87706 Nck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87707 Shc SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87708 Ras-GAP SH2 Domain Phosphotyrosine Capture Kit

6pull-downs $408

87709 Shp2 SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87710 PLC SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87711 Lyn SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87712 Hck SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87713 FGR SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87714 Cbl SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87715 Syk SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

87716 PI3K SH2 Domain Phosphotyrosine Capture Kit 6pull-downs $408

Phosphoprotein Pull-Down with SH2 Domains


Use of Titanium Dioxide for Phosphopeptide Enrichment

TitaniumdioxideenrichmentofphosphopeptidesamplesisanessentialtoolforcomprehensiveMSanalysisofphosphopeptides.TheuniquephosphopeptidebindingpropertiesofTiO2resinresultsindifferentselectivityandpreferentialenrichmentofmultiplyphosphorylatedpeptidescomparedtoimmobilizedchelatedmetalions(IMAC).TiO2resintypicallyenrichesmorephosphopeptidesthanIMACresins,andTiO2preferentiallyenrichesmonophos-phorylatedpeptides.

TiO2resintypicallybindsphosphopeptideswithahigheraffinitythanIMACandthepreferentialenrichmentofsinglyphosphory-latedpeptideswithTiO2islikelyduetodifficultyinelutingmultiplyphosphorylatedpeptides.Toenrichamorecomprehensivesetofphosphopeptides,theflow-throughandwashesfromIMACresincanbeacidifiedandappliedtoTiO2resinsandtheresultingelu-atespooledforanalysis.Inaddition,thenumberofphosphopep-tidesrecoveredcanbesignificantlyincreasedbypre-fractionationofsampleswithstrongcationexchangechromatographytoreducesamplecomplexitypriortoenrichmentwithTiO2orIMACprocessing.

Thermo Scientific Pierce TiO2 Phosphopeptide Enrichment and Clean-up KitSelective enrichment and Clean-up of phosphopeptides in under 2 hours.

ThePierceTiO2PhosphopeptideEnrichmentandClean-upKitenablesfast,selectiveenrichmentofphosphorylatedpeptides.Thecompletekitincludes24titaniumdioxide(TiO2)spintipsandgraphitespincolumnswithbufferstofacilitatepreparationofenrichedanddesaltedphosphopeptidesforanalysisbymassspectrometry(MS).

Highlights:• Convenient–spin-columnformatofTiO2andgraphitecolumns

enableparallelprocessingandcleanupofmultiplesamplesinlessthan2hours

• High capacity–eachcolumnenrichesupto300μgofphosphopeptides

• Complementary–TiO2enrichesauniquesetofphosphopeptidesthatcomplementsthePierceFe-NTAIMACPhosphopeptideEnrichmentKit

•High selectivity–recoverphosphopeptideswith>90%selectivity

ThePierceTiO2PhosphopeptideEnrichmentandClean-upKitprotocolincludesstringentwashingconditionsthatincreasetheselectivityforphosphopeptidesto>95%.Becausesomeofthesaltsinthesestringentwashesmaystillbepresentintheelutedsamples,thePierceTiO2PhosphopeptideEnrichmentandClean-upKitincludesgraphitespincolumnstodesaltandconcentrateenrichedphosphopeptides,resultinginmoresuccessfulphos-phopeptideanalysisresults.Thiskitiscompatiblewithsamplesdigestedinsolutionorafterin-geldigestionwithtrypsinorotherMSgradeproteases.UsingthePierceTiO2PhosphopeptideEnrichmentandClean-upKitandthePierceFe-NTAIMACPhosphopeptideEnrichmentKitswillenablecomplementarysetsofphosphopeptidestoberemovedfromcomplexsamples.

Number of Phosphates per Pepti0de

Resin 1 2 3 4 5 6 Total

TiO2 492 103 8 4 0 1 608

Fe-NTA IMAC 234 34 216 3 1 0 488

Overlap 155 0 1 0 0 0 156

Selective enrichment of singly and multiply phosphorylated phosphopeptides with Thermo Scientific Pierce TiO2 and FE-NTA IMAC resins. Averagephos-phopeptideenrichmentresultsfromduplicateexperimentsshowingthenumberofphosphopeptidescontainingoneormorephosphateperpeptideenrichedusingeitherresin.PeptidespectrumsummaryresultswereexportedfromProteomeSoftwareScaffold3.0.

120

100

80

60

40

20

Total PhosphopeptidesUnique Phosphopeptides% Acidic Residues

0

TiO2 Fe-NTA

Phos

phop

eptid

es (%

)

961 240

998238

TiO2 selectively enriches more phosphopeptides than Fe-NTA IMAC.Thenum-bersrefertothetotalanduniquenumberofphosphopeptidesenrichedbyeachmethod.TheY-axisisthepercentageofphosphopeptidesinthenumberoftotalanduniquepeptides,orthepercentageofacidicresiduesinenricheduniquephosphopeptides.

References:LarsenM.R.,et al.(2005).Highlyselectiveenrichmentofphosphorylatedpeptidesfrompeptidemixturesusingtitaniumdioxidemicrocolumns.Mol Cell Proteomics. 4(7):873-86.Carrascal,M.,et al.(2008).PhosphorylationAnalysisofPrimaryHumanTLymphocytesUsingSequentialIMACandTitaniumOxideEnrichment.J. Proteome Res.7(12):5167-5176.Wilson-Grady,J.T., et al.(2008).PhosphoproteomeAnalysisofFissionYeast.J. Proteome Res. 7(3):1088-1097.Larsen,M.R.,et al.(2004).Improveddetectionofhydrophilicphosphopeptidesusinggraphitepowdermicrocolumnsandmassspectrometry:evidenceforinvivodoublyphos-phorylateddynaminIanddynaminIII.Mol. Cell Proteomics.3:456-465.


Description

Pkg. Size

U.S. Price

88301 Pierce TiO2 Phosphopeptide Enrichment and Clean-up Kit Sufficient For: 24 phosphopeptide enrichment and cleanup reactions. KitContents:TiO2SpinTips,24tips

TrifluoroaceticAcid,1mL90%LacticAcid,2mLPyrrolidine,200μLCentrifugeColumnAdaptors,24adaptorsPierceGraphiteSpinColumns,24columns

24-RxnKit $375

Phosphopeptide Enrichment



Thermo Scientific Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment KitTiO2 magnetic particles for high throughput phosphopeptide isolation.

ThePierceMagneticTitaniumDioxidePhosphopeptideEnrichmentKitisforisolatingphosphopeptidesfromcomplexbiologicalsam-plesusingtitaniumdioxide-coatedmagneticbeads.TheTiO2ligandselectivelybindspeptidescon-tainingphosphorylatedserine(Ser),tyrosine(Tyr)orthreonine(Thr),enablingphosphopeptideenrichmentfromprotease-digestedsamples.Theisolatedphos-phopeptidesarecompatibleforanalysisdownstreambymassspectrometry(Table2).

Thehigh-performance,ironoxide,superparamagneticparticlesarevalidatedandoptimizedforusewithhigh-throughputmagneticplatforms,suchastheThermoScientificKingFisher96andKingFisher®FlexInstruments.Thebeadsalsoenablepremiumperformanceforsimplebenchtopapplicationsusinganappropriatemagneticstand.

Highlights:• Complete MS-compatible Kits–includeready-to-usebinding,

washandelutionbuffersthatareoptimizedforphosphopeptideenrichmentanddownstreamanalysisbyMALDIandESImassspectrometry

• Optimized for HTS–procedurevalidatedforprocessing1to96samplesatatime;completeentireassayinabout15minutesusingaKingFisherFlexInstrument

• Stable affinity ligand–titaniumdioxideisspeciallycoatedasafilmonthemagneticparticles

• Selective–affinitysystemisselectiveforphosphorylatedSer,TyrandThr;exhibitsminimalnon-specificbindingtoacidicresidues

• Sensitive–affinityprovidesmorethan1000timesgreatersensi-tivitythantraditionalIMACtechnologies;enablesenrichmentandMS-measurementoflessthan100fmolofphosphoprotein

Table 2. Phosphopeptide enrichment improves MS-identification of phospho-proteins. Twomilligramsofatrypticdigestpreparedfromperiferalbloodmono-nuclearcells(lymphocytes)withandwithoutphosphopeptideenrichmentwereanalyzedbyMS.EnrichmentwasperformedwiththeThermoScientificPierceTitaniumDioxidePhosphopeptideEnrichmentKitusingtheThermoScientificKingFisher96Instrument.SampleswereanalyzedonaThermoScientificLTQOrbitrapMassSpectrometer.

Enriched Non-Enriched

Totalnumberofproteinsidentified 185 247

Totalnumberofphosphoproteinsidentified 160 1

Totalnumberofpeptidesidentified 2347 2457

Totalnumberofphosphopeptidesidentified 2009 7

Totalnumberofuniquephosphopeptidesidentified 177 1

Relativeenrichmentforphosphopeptides(%) 86 0.3


Description

Pkg. Size

U.S. Price

88811 Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment KitIncludes:TiO2MagneticBeads(20X)

BindingBufferWashingBufferElutionBufferThermo-Fast96RoboticPCRPlate(0.2mLwells)

Kit1mL100mL25mL3mL2plates

$427

88812 Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit, Trial SizeIncludes:TiO2MagneticBeads(20X)

BindingBufferWashingBufferElutionBufferThermo-Fast96RoboticPCRPlate(0.2mLwells)

Kit0.25mL100mL25mL3mL2plates

$240


Thermo Scientific Pierce Fe-NTA Phosphopeptide Enrichment KitNew Fe-NTA format optimized for capture and recovery of phosphopeptides.

ThenewPierceFe-NTAPhosphopeptideEnrichmentKitenablesfastandefficientenrichmentofphosphorylatedpeptides.Thesespincolumnsareeasytouseandrequirelessthan1hourtoprocessproteindigestsorstrongcation-exchangepeptidefractionsforanalysisbymassspectrometry(MS).

Highlights:•Convenientspinformatforparallelprocessingof

multiplesamples•High-bindingcapacityresinforenrichingupto150μg

ofphosphopeptidespercolumn•Excellentenrichmentandrecoveryofphosphopeptides

Proteinphosphorylationisessentialtobiologicalfunctions,includingcellsignaling,growth,differentiation,divisionandprogrammedcelldeath.Over500proteinkinasescatalyzephosphorylationofspecifictargets,primarilyonserine,threonine,andtyrosineresidues.

Massspectrometryisincreasinglybeingusedtoidentifyandquantifyphosphorylationchanges;however,phosphoproteinandphosphopeptideanalysisbyMSislimitedbymanyfactors,includingdigestionefficiency,lowstoiochiometry,lowabundance,hydrophilicity,poorionizationandpoorfragmentation.Asaresult,phosphopeptideenrichmentisessentialtosuccessfulMSanaly-sis.ThenewPierceFe-NTAPhosphopeptideEnrichmentKitiscompatiblewithourlysis,reduction,alkylation,anddigestionreagentsandwithThermoScientificPierceGraphiteSpinColumnstoprovideacompleteworkflowforphosphopeptideenrichment.

Toassessphosphopeptideenrichmentfromlysates,culturedU2-OScellsarrestedwithnocodazole(100ng/mL,25hours)werelysedwith6Mureain50mMTris,pH8.0containingThermoScientificHaltProteaseandPhosphataseInhibitorCocktail(Product#78440).ProteinconcentrationwasdeterminedwithThermoScientificPierce660nmProteinAssay(Product#22660).ProteinswerereducedwithThermoScientificBond-BreakerTCEPSolution,NeutralpH(Product#77720),alkylatedwithsingle-useiodoacetamide,(Product#90034)digestedovernightwithMS-gradetrypsin(Product#90055),anddesaltedwithThermoScientificHyperSep-C18Cartridges(Product#60108-305).Anequivalentof200μgofpeptidesweredriedanddissolvedin5%aceticacidorSigmaPhos-Select™Buffer.PhosphopeptideswereenrichedwithPierceFe-NTAPhosphopeptideEnrichmentKitorSigmaPhos-SelectReagentsandthendesaltedandconcentratedwithPierceGraphiteSpinColumns(Product#88302)accordingtoinstructions.

EnrichedphosphopeptidesampleswereanalyzedbyLC-MS/MS.ANanoLC™-2DHPLC(Eksigent)withaProteoPepIIC18Column(75μmIDx20cm,NewObjective)wasusedtoseparatepeptidesusinga4-40%gradientofsolvents(A:water,0.1%formicacid;B:acetonitrile,0.1%formicacid)at250nLperminutefor60minutes.PeptideswereidentifiedwithaThermoScientificLTQOrbitrapXLETDMassSpectrometerusingatopfourexperimentconsistingofhigh-resolutionMSfollowedbyacquisitionoffourMS/MSspectrausingtheCIDmodeoffragmentation.LC-MS/MSdatawereinterpretedwithMascot2.2(MatrixScience)andScaffold2.6(ProteomeSoftware).

ToachieverobustMSresults,enrichmentofphosphopeptidesamplesisessentialbecauseoflowstoichiometryandabundanceandpoorionizationrelativetononphosphorylatedpeptides.Wehavedevelopedanefficientmeanstoenrichphosphopeptidesfromcomplexsamples.ThenewThermoScientificPierceFe-NTASpinColumnseffectivelycapture,enrich,andrecoverphosphopeptides.Thesecolumnsenrichahigherpercentageofphosphopeptidesthanotherresinsandwithanoverallhighernumberoftotalanduniquephosphopeptides(Figure8andTable3).

0

10

20

30

40

50

60

70

Phos

phop

eptid

es (%

)

Thermo Scientific Pierce Fe-NTA Kit Sigma Phos-SelectReagent

Total Phosphopeptides

862

43090

178Unique Phosphopeptides

Figure 8. Our kit enriched a greater percentage of total and unique phospho-peptides from U2-OS cell lysate.Numbersrefertothetotalanduniquenum-berofphosphopeptidesidentifiedineachcondition.AsummaryofresultsislistedinTable3.


Table 3. Average phosphopeptide enrichment results from duplicate experiments.§

Thermo Scientific Pierce Fe-NTA

Sigma Phos-Select

Totalphosphopeptides 862 430

Totalpeptides 1,753 1,665

Totaluniquepeptides 393 395

Totaluniquephosphopeptides 178 90

Totalphosphopeptides(%) 53 31

Uniquephosphopeptides(%) 50 27.5

§ Peptide summary results were exported from Scaffold and analyzed and summarized with Microsoft Excel® and Access®.

Multiplephosphorylatedaminoacidswithinapeptidecontributetothecomplexityofphosphopeptideanalysis.PierceFe-NTASpinColumnsenrichpeptideswiththreeormorephosphorylatedsitesandsignificantlyoutperformothercommerciallyavailablecolumns(Figure9).Phosphopeptideenrichmentgreatlyreducessamplecomplexityandenableseffectiveidentificationandcharacteriza-tionofphosphorylatedpeptidesbyMS(Figure10).

0

20

40

60

80

100

120

Phos

phop

eptid

es (%

)

Single Double

Phosphates per Peptide

Triple ≥4

Thermo Scientific Pierce Fe-NTA Kit110

29

132 12 1 0

Sigma Phos-Select Reagent

Figure 9. The Thermo Scientific Pierce Fe-NTA Phosphopeptide Enrichment Kit effectively captures phosphopeptides with multiple phosphates.

ThePierceFe-NTAPhosphopeptideEnrichmentKitcontainsdetailedinstructionsandallnecessarycomponentstoload,washandelutephosphopeptideswithinanhour.Thiskitiscompatiblewithsamplesdigestedinsolutionorafterin-geldigestionusingtheThermoScientificIn-gelTrypticDigestionKit(Product#89871).

a11+2H+1

y2 b4y3 b5y4 b6 y5 b7y6

b8y7 y8b9 y9b10

G E P N V S Y+80 I C+57 S RR S C+57 I Y+80 S V N P E G

m/z

0%

25%

50%

75%

100%A.

B.

Rela

tive

Inte

nsity

0 250 500 750 1000 1250

m/z

0%

25%

50%

75%

100%

Rela

tive

Inte

nsity

0 250 500 750 1000 1250

GSK3A_HUMANGlycogen Synthase Kinase-3 alpha

(R)GEPNVSyICSR(Y)

CDC2_HUMANCell division control protein 2 homolog

(K)IGEGTyGVVYK(G)

b3y2

b4y3

b5 y4 y5 b6 b7y6

b8 y7 b9

y8

y9b10y10

I G E G T Y+80 G V V Y KK Y V V G Y+80 T G E G I


Description

Pkg. Size

U.S. Price

88300 Pierce Fe-NTA Phosphopeptide Enrichment KitSufficient for 30 samples

Kit $279

Related Products88302 Pierce Graphite Spin Columns 30columns $105

Figure 10. Example MS/MS spectra from enriched phosphopeptides. Lowercaselettersindicatethepositionoftyrosinephosphorylationinenrichedpeptides.Panel A:Glycogensynthase-3alpha.Panel B:cdc2/cyclindependentkinase1.



Cell Surface Protein Isolation Kit

Convenient biotinylation and isolation of cell surface proteins for Western blot analysis.

TheThermoScientificPierceCellSurfaceProteinIsolationKitisacompletekitfortheconvenientbiotinylationandisolationofmammaliancellsurfaceproteins,specificallytargetingcellsurfaceproteinstotheexclusionofintracellularproteins.Thekitefficientlylabelsproteinswithaccessiblelysineresiduesandsufficientextracellularexposure.

Theisolationprocedureusesacell-impermeable,cleavablebiotinylationreagent(Sulfo-NHS-SS-Biotin)tolabelsurfaceproteinsatexposedprimaryamines.Cellsarethenharvestedandlysed,andthelabeledsurfaceproteinsareaffinity-purifiedusingThermoScientificNeutrAvidinAgaroseResin.Theisolatedcellsurfaceproteinscontainasmall,nonreactivetagoftheoriginallylabeledprimaryaminesbutarenolongerbiotinylated(biotinremainsboundtotheresin).

Highlights:• Isolates cell surface proteins–reducescomplexityoftotal

cellularprotein• Efficiently recovers labeled proteins–cleavablebiotinallowsfor

nearly100%recoveryofisolatedcellsurfaceproteins• Convenience–allreagentsareprovidedinonekit,alongwith

completeinstructionsforlabeling,celllysisandpurificationofcellsurfacemembraneproteins

• Western blotting applications–proteinsrecoveredinSDS-PAGEbufferareloadeddirectlyontopolyacrylamidegels

• Robust system–protocoldesignedfordiversecelllines,includingNIH3T3,HeLa,C6andA431

A. Cell Surface Proteins

EGFR

IGF-1Rβ

Hsp90 Calnexin

Integrin α5

Integrin β1

R F E R F E

R F E R F E

R F E R F E R F E R F E

R F E R F E

R F E R F E

B. Intracellular Proteins

+ – + –

+ –

+ –

+ –

+ –

Protein isolation is specific to cell surface proteins. PanelsareWesternblotresultsforknowncellsurfaceproteins (Panel A) andintracellularproteins(Panel B)fromHeLacellstestedwiththeCellSurfaceProteinIsolationKit.Plussymbol(+)denotesresultsforcellstreatedwiththeSulfo-NHS-SS-Biotinreagent;minussymbol(-)denotesresultsforcellsthatwerenottreatedwiththebiotinreagentbutwereotherwisecarriedthroughthekitprocedure.Lanesareno-sampleresin-control(R),flow-through(F)andeluted(E)frac-tions.Presenceoftargetcellsurfaceproteinsintheplus-Eandminus-Fconditionsindicatesuccessfulisolationwiththekit.PresenceofintracellularproteinsinFconditionofbothplusandminusconditionsindicatesthatthelabelingandpurificationprocedureisspecifictocellsurfaceproteins.



89881 Cell Surface Protein Isolation KitSufficient reagents and accessories for eight experiments, each involving four T75 flasks of confluent cells.Includes:EZ-Link®Sulfo-NHS-SS-Biotin

QuenchingSolutionLysisBufferNeutrAvidinAgaroseWashBufferDithiothreitol(DTT)BupHPhosphateBufferedSalineBupHTrisBufferedSalineSpinColumnsandAccessories

Kit

8x12mgvials16mL4.5mL2.25mL34mL8x7.7mgmicrotubes2packs1pack

$348

Biotinylate cells

30 minutes at 4˚C

Transfer cell pellet to 1.5 ml tube

Lyse cells 30 minutes on ice

Isolate biotinylated proteins on NeutrAvidin® Agarose Resin

Perform electrophoresis

or other application

Wash gel then elute with SDS-PAGE sample buffer + 50 mM DTT

Harvest Cells

Quench Reaction

gel

B

SH

+

protein – SH

N

N

gel

N

B

S-S-protein

N

N Biotin NeutrAvidinBiotin-Binding Protein

1D gel

B

45

40

35

30

25

20

15

107.55.0

Protocol summary for the Thermo Scientific Pierce Cell Surface Protein Isolation Kit.

Protein Enrichment


Glycoprotein Isolation Kits

Isolate glycoproteins from complex protein mixtures.

Twolectin-basedThermoScientificGlycoproteinIsolationKits,concanavalinA(ConA)andwheatgermagglutinin(WGA),allowisolationofglycoproteinsfromcomplexproteinmixtures,includingserum,tissueandculturedcelllysates,thusenablingdownstreamanalysis.ConAlectinrecognizesa-linkedmannoseandterminalglucoseresidues,whileWGAlectinselectivelybindstoN-acetylglucosamine(GlcNAc)groupsandtosialicacid.

Highlights:• High recovery–equivalentorgreaterglycoproteinrecoveryvs.

competitorkitsandlectinresins• Fast–glycoproteinpurificationinlessthanonehour• Versatile–isolateglycoproteinsfromvarioussampletypes;

e.g.,humanserumandcelllysate• Robust–lectindoesnotleachfromresinwhen

processingsample• Convenient–completekitcontainslectinresinsandspin

columnswithallnecessaryreagents• Compatible with Bradford-based protein assays–dialysisor

proteinprecipitationofrecoveredglycoproteinsisnotrequiredbeforeproteinassay

Ther

mo

Scie

ntifi

c

Ther

mo

Scie

ntifi

c

Supp

lier A

Supp

lier A

Supp

lier G

ConA

Supp

lier G

ConA

B.A.

Human Serum CHO Lysate

Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using ConA resin. HumanserumandCHOlysatesampleswereprocessedwiththeThermoScientificGlycoproteinIsolationKit,ConAandwithothercommerciallyavailableConAresins.Anequivalentamountoftotalproteinwasappliedtoeachresin.ElutedglycoproteinfractionswerecomparedwithConAResinboiledinSDS-PAGEBuffertoreleaselectins.Allfractionswerenormalizedbyvolumeandresolvedon8-16%polyacrylamidegels.Gelsweresilver-stained.A.ElutedglycoproteinfractionsfromappliedhumanserumandB.elutedglycoproteinfractionsfromappliedCHOlysate.ArrowsidentifyproteinbandsthatresultfromConAleachingfromtheresinduringtheelutionprocess.

Com

petit

or A

Supp

lier A

Supp

lier A

B.A.

CHO LysateHuman Serum

Ther

mo

Scie

ntifi

c

Ther

mo

Scie

ntifi

c

Glycoprotein isolation from human serum and cell lysate: performance comparison of kits using WGA resin.HumanserumandCHOlysatesampleswereprocessedwiththeThermoScientificGlycoproteinIsolationKit,WGAandwithothercommerciallyavailableWGAresins.Anequivalentamountoftotalproteinwasappliedtoeachresin.Elutedglycoproteinfractionswerenormalizedbyvolumeandresolvedon8-16%polyacrylamidegels.A.ElutedglycoproteinfractionfromappliedhumanserumandB.elutedglycoproteinfractionfromappliedCHOlysate.


Description

Pkg. Size

U.S. Price

89804

Glycoprotein Isolation Kit, ConASufficient reagents to isolate glycoproteins with strong affinity for ConA from 10 samples of up to 640µL (1-1.5mg total protein) each.Includes:ConALectinResin,1.1mLresinsuppliedas

a50%slurryBinding/WashBuffer,5XStockSolutionElutionBufferColumnAccessoryPack,10SpinColumnswithCapsand20CollectionTubes

Kit

6.5mL5mL

$223

89805 Glycoprotein Isolation Kit, WGASufficient reagents to isolate glycoproteins with strong affinity for WGA from 10 samples of up to 640µL (1-1.5mg total protein) each.Includes:WGALectinResin,1.1mLresinsuppliedasa

50%slurryBinding/WashBuffer,5XStockSolutionElutionBufferColumnAccessoryPack,10SpinColumnswithCapsand20CollectionTubes

Kit

6.5mL5mL

$282

Protein Enrichment


Ubiquitin Enrichment Kit

Recover ubiquitin modified protein in <45 minutes.

Theubiquitinproteasomepathwayistheprincipalnon-lysosomalpathwaythatcontrolstheproteolysisofproteins.Thispathwayissignificantlyinvolvedinavarietyofcellularprocesses,includingDNArepair,transcriptionalregulation,signaltransduction,cellmetabolismandmorphogenesis.Differencesintotalubiquitinationortheubiquitinationofspecificproteinsaffectnumerouspathologicalconditions,includingmalignancies,certaingeneticdiseasesandneurodegenerativediseases.1

TheThermoScientificUbiquitinEnrichmentKitisolatespolyubiquitinproteinconjugatesfromculturedcellsandtissuesamples.TheenrichedfractionisanalyzedtodeterminetheamountofgeneralubiquitinconjugatespresentortoidentifyaspecificproteinbyWesternblotting.Theassayprotocolisfast,straightforwardandallowsisolationofpolyubiquitinatedproteinsandthefractionationofmonoubiquitinatedspeciesintheresinflow-through.TheUbiquitinEnrichmentKitoutperformsothersuppliers’kitsandprovidesacleanandspecificpreparationofproteinswhencomparedtoacontrolresin.

Highlights:•Fast–lessthan45minuteshands-ontime• Complete–includesallreagentsneededforubiquitin-modified

proteinenrichmentfromculturedcellsandtissuesamples,includingspincolumnsandubiquitinantibody

• Flexible–sampleincubationfrom2hourstoovernightallowsassaytobecompletedinseveralhoursorinlessthan30minutesafterovernightincubation

• Robust–compatiblewithallThermoScientificCellLysisSolutionsandstandardRIPAformulations

• Multiple-sample format–easilyprocesses1-15samplesconcurrently

20

40

5060

80100120

220

Thermo Scientific

KitSupplier

CAb-basedMethod

GSH Resin

H F E F E F E F E

kDa

The Thermo Scientific Ubiquitin Enrichment Kit recovers more ubiquitin-modified proteins than any other method. Epoxomicin-treatedHeLacelllysates(EHeLa,150μg)wereenriched.Afterelution,allsampleswerenormalizedtotheinitialload(EHeLaload).Theflow-throughandelutionobtainedusingtheThermoScientificUbiquitinEnrichmentKitareshownfirst.Theresultsusinganothersupplier’senrichmentkitareshownforcomparison(SupplierC,manufacturer’sinstructionsforthiskitwerefollowed).Additionally,theresultsobtainedusingananti-ubiquitinmonoclonalantibody-basedenrichmentscheme(antibody-based)andanegativecontrolresin(GSHresin)areshown.Theelutionfromeachresinshowstheamountofubiquitin-modifiedproteinthatwascapturedusingthatmethod.

Reference1.Ciechanover,A.(1998).Theubiquitin-proteasomepathway:onproteindeathandcelllife.

EMBO J.17(24),7151-1760.



89899

Ubiquitin Enrichment KitContains sufficient materials for enriching up to 15 lysate samples containing ~0.15mg total protein per sample.Pack 1PolyubiquitinPositiveControl(1,000X),50μL,2mg/mLAnti-ubiquitinAntibody,50μLrabbitantiserumPack 2PolyubiquitinAffinityResin,300μL,suppliedasa25%slurryBindingCapacity:~1μgper20μLofslurryBupHTrisBufferedSalinePack,1ea.,makes500mLof0.025MTris,0.15MNaCl;pH7.2SpinColumnsandAccessories,18columnswithtopandbottomcaps

Kit $399


Antibody Purification Overview

Antibodies specific for an antigen of interest are one of the most useful and important tools that biology researchers can possess. The production and use of specific antibodies as detection probes and purification ligands (i.e., immunotechnology) has revolutionized bioresearch and diagnostic technologies. Animalsimmunizedwithpreparedantigenswillproducespecificantibodiesagainsttheantigen.Whenpurifiedfromserumorhybridomacelllinesthatarepreparedfromtissueoftheimmunizedanimal,theantibodycanbeuseddirectly(orafterlabelingwithenzymeorfluorescenttags)toprobethespecificantigeninWesternblotting,ELISAoravarietyofotherapplications.Antibodiesaremostcommonlypurifiedbyoneoftwoaffinitypurificationmethods:generalimmunoglobulinpurification(pages58-65)orspecificantibodypurification(seepages26-35).SeealsoTable1.

General Purification of Immunoglobulins

Becauseantibodieshavepredictablestructure,includingrelativelyinvariantdomains,ithasbeenpossibletoidentifycertainproteinligandsthatarecapableofbindinggenerallytoantibodies,regardlessoftheantibody’sspecificitytoantigen.ProteinA,ProteinGandProteinLarethreebacterialproteinswhoseantibody-bindingpropertieshavebeenwellcharacterized.Theseproteinshavebeenproducedrecombinantlyandusedroutinelyforaffinitypurificationofkeyantibodytypesfromavarietyofspecies.AgeneticallyengineeredrecombinantformofProteinAandG,calledProteinA/G,isalsoavailable.Theseantibody-bindingproteinsareavailableimmobilizedtobeadedagaroseresin,ThermoScientificUltraLinkBiosupportandcoatedontomicroplates.

ProteinsA,G,A/GandLbindtoantibodiesatsitesotherthantheantigen-bindingdomain.Therefore,theseproteinscanbeusedinpurificationschemessuchasimmunoprecipitation.

ProteinsA,G,A/GandLhaveuniqueproperties,whichmakeeachonesuitablefordifferenttypesofantibodytargets(e.g.,antibodysubclassoranimalspecies).ItisimportanttorealizethatuseofProteinA,GorLresultsinpurificationofgeneralimmunoglobulinfromacrudesample.Dependingonthesamplesource,antigen-specificantibodymayaccountforonlyasmallportionofthetotalimmunoglobulininthesample.Forexample,generallyonly2-5%oftotalIgGinmouseserumisspecificfortheantigenusedtoimmunizetheanimal.

Table 1. Antibody Purification Methods.

Purification Type Description Available Thermo Scientific Support

General Negativeselection Removalofallnon-immunoglobulinproteinsfromaserumsample

MelonGel

IgGenrichment ImmobilizedglobulinbindingproteinstoselectivelyremoveIgGfromaserumsample

ImmobilizedProteinA

ImmobilizedProteinG

ImmobilizedProteinA/G

ImmobilizedProteinL

IgGenrichment Thiophilicadsorption ThiophilicAdsorbent

IgMenrichment UseMannanbindingproteintoselectivelyisolateIgM ImmobilizedMannanBindingProtein

IgAenrichment UseJacalin,aD-galactosebindinglectin,toselectivelyisolateIgA

ImmobilizedJacalin

IgYenrichment Delipidationandprecipitationfromeggyolks IgYPrecipitationReagent

Specific (seepage26)

Affinitypurification Createanaffinitycolumnwiththeantigenusedtoimmunizetheanimal.

NHS-esterActivatedAgarose-forimmobilizingantigensviaprimaryamines

AminoLinkPlusAgarose-forimmobilizingantigensviaprimaryamines

SulfoLinkResin-forimmobilizingantigensviareducedsulfhydryls

CarboxyLinkResin-forimmobilizingantigensviacarboxylicacids

Antibody Purification


w = weak binding, m = medium binding, s = strong binding, nb = no binding, – means information not available * Data represent a summary of binding properties reported in the literature. Inevitably some discrepancies exist among reported values as a result of differences in binding

buffer conditions and form of the proteins used. † Binding will occur only if the appropriate kappa light chains are present. Antibodies lambda light chains will not bind, regardless of their class and subclass.

Protein A Protein G Protein A/G Protein L†ThiophilicAdsorbent

HumanIgG s s s s m

MouseIgG s s s s s

RabbitIgG s s s w m

GoatIgG w s s nb s

RatIgG w m m s s

SheepIgG w s s nb s

CowIgG w s s nb s

GuineaPigIgG s w s – s

HamsterIgG m m m s –

PigIgG s w s s s

HorseIgG w s s – s

DonkeyIgG m s s – –

DogIgG s w s – s

CatIgG s w s – s

MonkeyIgG(Rhesus)

s s s – s

ChickenIgY nb nb nb nb m

HumanIgM w nb w s m

HumanIgE m nb m s –

HumanIgD nb nb nb s –

HumanIgA w nb w s m

HumanIgA1 w nb w s m

HumanIgA2 w nb w s m

HumanIgG1 s s s s m

Protein A Protein G Protein A/G Protein L†ThiophilicAdsorbent

HumanIgG2 s s s s m

HumanIgG3 w s s s m

HumanIgG4 s s s s m

HumanFab w w w s m

HumanScFv w nb w s m

MouseIgG1 w m m s s

MouseIgG2a s s s s s

MouseIgG2b s s s s s

MouseIgG3 s s s s s

RatIgG1 w m m s s

RatIgG2a nb s s s s

RatIgG2b nb w w s s

RatIgG2c s s s s s

CowIgG1 w s s nb s

CowIgG2 s s s nb s

SheepIgG1 w s s nb s

SheepIgG2 s s s nb s

GoatIgG1 w s s nb s

GoatIgG2 s s s nb s

HorseIgG(ab) w nb w – s

HorseIgG(c) w nb w – s

HorseIgG(T) nb s s – s

MouseIgM nb nb nb s m

Immobilized Protein L, Protein A, Protein G and Protein A/G

Weofferthesepopularantibody-bindingproteinsimmobilizedonseveraldifferentresins,beadsandplatesforuseinimmunoaffinitypurificationtechniques.Allfourproteins(A,G,A/GandL)areavailableaspurifiedrecombinantsimmobilizedtocrosslinked6%beadedagarose.Thisisthetraditionalformathistoricallyusedforsmall-scalecolumnpurificationandimmunoprecipitationmethods.Ouragaroseresinsdifferfromthosetypicallyofferedbyothersuppliersinthatourimmobilizationmethodismorestableandresultsinlessnonspecificbinding.Wealsooffer“Plus”versionsoftheProteinA,G,A/GandLagaroseresins,whichcontaintwice

theamountofproteinpermilliliterofresinandprovidefornearlytwicetheantibodybindingcapacity.

ProteinA,G,A/GandLarealsoavailableimmobilizedtoUltraLink®Biosupport,anextremelydurable,polyacrylamide-basedresinwithverylownonspecificbindingcharacteristics.TheUltraLinkFormatisaperfectsupportforworkingwithlargevolumesamplesinlarge-scalepurificationmethodsrequiringfastflowandhighpressure.

TheinteractionbetweenthevariousproteinsandIgGisnotequivalentforallspeciesorallantibodysubclasses.Thetablesonthefollowingpagewillhelpyoudecidewhichaffinityproteinisbestforyourapplication(Tables2and3).

Table 2. Characteristics of immunoglobulin-binding proteins.

Recombinant Protein L

Native Protein A

Recombinant Protein A

Recombinant Protein G

Recombinant Protein A/G

ProductionSource E. coli S. aureus E. coli E. coli E. coli

MolecularWeight 35,800 46,700 44,600 21,600 50,460

NumberofBindingSitesforIgG 4 4 4 2 6

Albumin-BindingSite No No No No No

OptimalBindingpH 7.5 8.2 8.2 5 5-8.2

Bindsto VLK FC FC FC FC

Table 3. Binding characteristics of immunoglobulin-binding proteins and Thermo Scientific Thiophilic Adsorbent.*


Protein A

Protein A Beads – Quick Reference

ProteinA NativeproteinpurifiedfromStaphylococcus aureus(46.7kDa;fourIgG-bindingsites)

Specificity(Table3)

BestforpolyclonalIgGfromrabbit,pig,dog,catserum;poorforMouseIgG1,humanIgG3,rat,goat,cow

SupportsOffered

Crosslinked6%beadedagaroseUltraLinkBiosupportTrisacrylGF-2000ResinMagneticparticles(1-4μm)

PackageFormats

Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAb™IgGPurificationKits(2sizes)

Storage 4°C,donotfreeze

Capacity ProteinA:12-19mghumanIgG/mLresinProteinAPlus:>35mghumanIgG/mLresin;

16-17mgmouseIgG/mLresinProteinATrisacrylResin:>15mghumanIgG/mLresinProteinAUltraLinkResin:>16mgofhumanIgG/mLresinProteinAPlusUltraLinkResin:>30mghumanIgG/mLresinProteinAMagnaBindBeads:>0.2mgrabbitIgG/mLbeadsProteinACoatedPlates:~4pmolrabbitIgG/wellRecombinantProteinA:≥12mghumanIgG/mLresinusingthe

IgGBufferSystem


Product #

Description

Pkg. Size

U.S. Price

20333 Protein A Agarose 5mL $ 337

20334 Protein A Agarose 25mL $1050

20356 Protein A Columns 5x1mL $ 385

44667 Protein A IgG Purification Kit Kit $ 515

20338 Protein A Trisacryl Resin 5mL $ 350

53139 Protein A UltraLink Resin 5mL $ 340

22810 Protein A Plus Agarose 1mL $ 120

22811 Protein A Plus Agarose 5mL $ 400

22812 Protein A Plus Agarose 25mL $1350

89924 Chromatography Cartridges, Protein A 2x1mL $ 170

89925 Chromatography Cartridge, Protein A 1x5mL $ 335

89952 NAb Protein A Plus Spin Columns 10x0.2mL $ 222

89956 NAb Protein A Plus Spin Columns 5x1mL $ 425

89960 NAb Protein A Plus Spin Column 1x5mL $ 400

89948 NAb Protein A Plus Spin Purification Kit Kit $ 270

89978 NAb Protein A Plus Spin Purification Kit Kit $ 265

53142 Protein A UltraLink Plus Resin 5mL $ 399

45202 Protein A Spin Plate 1plate $ 188

21348 MagnaBind Protein A Beads 5mL $ 325

15130 Protein A, Clear 96-Well Plates 5plates $ 160

15132 Protein A, Clear 8-Well Strip Plates 5plates $ 178

15154 Protein A, White 96-Well Plates 5plates $ 178

15155 Protein A, Black 96-Well Plates 5plates $ 178

20365 Recombinant Protein A Agarose 5mL $ 275

20366 Recombinant Protein A Agarose 25mL $1001

Protein G

Protein G Beads – Quick Reference

ProteinG RecombinantproteinexpressedinE. coli(21.6kDa;twoIgGbindingsites)

Specificity(Table3)

BestforIgGfrommouse,human,cow,goatandsheep;poorforguineapig,pig,dogandcat

SupportsOffered

Crosslinked6%beadedagaroseUltraLinkBiosupportMagneticparticles(1-4μm)

PackageFormats

Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NabIgGPurificationKits(2sizes)


Capacity ProteinG:11-15mghumanIgG/mLresinProteinGUltraLinkResin:>20mgofhumanIgG/mLresinProteinGPlus:>20mghumanIgG/mLresinProteinGUltraLinkResin:>20mgofhumanIgG/mLresinProteinGPlusUltraLinkResin:>25mghumanIgG/mLresinMagnaBindProteinGBeads:>0.2mgrabbitIgG/mLbeadsProteinGCoatedPlates:~2pmolrabbitIgG/wellProteinGPlus:>20mghumanIgG/mLresinProteinGPlusUltraLinkResin:>25mghumanIgG/mLresin


Product #

Description

Pkg. Size

U.S. Price

20398 Protein G Agarose 2mL $ 212

20399 Protein G Agarose 10mL $ 760

20397 Protein G Agarose 25mL $1654

89926 Pierce Chromatography Cartridges, Protein G 2x1mL $ 190

89927 Pierce Chromatography Cartridge, Protein G 1x5mL $ 390

89953 NAb Protein G Spin Columns 10x0.2mL $ 251

89957 NAb Protein G Spin Columns 5x1mL $ 455

89961 NAb Protein G Spin Columns 1x5mL $ 425

89949 NAb Protein G Spin Purification Kit Kit $ 310

89979 NAb Protein G Spin Purification Kit Kit $ 368

21193 Pierce Recombinant Protein G 5mg $ 300

53125 Protein G UltraLink Resin 2mL $ 270

53126 Protein G UltraLink Resin 10mL $ 910

53127 Protein G UltraLink Columns 2x2mL $ 540

45204 Protein G Spin Plate 1plate $ 209

22851 Protein G Plus Agarose 2mL $ 260

22852 Protein G Plus Agarose 10mL $ 895

53128 Protein G Plus UltraLink Resin 2mL $ 335

21349 MagnaBind Protein G Beads 5mL $ 325

15131 Protein G, Clear 96-Well Plates 5plates $ 178

15133 Protein G, Clear 8-Well Strip Plates 5plates $ 195

15156 Protein G, White 96-Well Plates 5plates $ 195

15157 Protein G, Black 96-Well Plates 5plates $ 195



Protein A/G

Protein A/G Beads – Quick Reference

ProteinA/G RecombinantproteinexpressedinE. coli(50.5kDa;sixIgG-bindingsites)

Specificity(Table3)

BestforpolyclonalIgGfrommanyspecies;poorforindividualsubclassesthathavehighestaffinityforProteinAorProteinG.

SupportsOffered

Crosslinked6%beadedagaroseUltraLinkBiosupport

PackageFormats

Resinslurries(3sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAbIgGPurificationKits(2sizes)


Capacity ProteinA/G:>7mghumanIgG/mLresinProteinA/GUltraLinkResin:>20mghumanIgG/mLresinProteinA/GPlus:>50mghumanIgG/mLresinProteinA/GPlusonUltraLinkSupport:>28mghumanIgG/mLresinProteinA/GCoatedPlates:~5pmolrabbitIgG/well


Description

Pkg. Size

U.S. Price

15138 Protein A/G, Clear 96-Well Plates 5plates $ 196

20421 Protein A/G Agarose 3mL $ 315

20422 Protein A/G Agarose 15mL $1150

89930 Pierce Chromatography Cartridges, Protein A/G 2x1mL $ 299

89931 Pierce Chromatography Cartridge, Protein A/G 1x5mL $ 399

89954 NAb Protein A/G Spin Columns 10x0.2mL $ 260

89958 NAb Protein A/G Spin Columns 5x1mL $ 460

89962 NAb Protein A/G Spin Column 1x5mL $ 435

89950 NAb Protein A/G Spin Purification Kit Kit $ 336

89980 NAb Protein A/G Spin Purification Kit Kit $ 375

53132 Protein A/G UltraLink Resin 2mL $ 270

53133 Protein A/G UltraLink Resin 10mL $ 910

21186 Pierce Recombinant Protein A/G 5mg $ 303

20423 Protein A/G Plus Agarose 2mL $ 340

53135 Protein A/G Plus on UltraLink Support 2mL $ 367

Protein L

Protein L Beads – Quick Reference

ProteinL RecombinantproteinexpressedinE. coli(35.8kDa;fourIgG-bindingsites)

Specificity(Table3)

Bestforhumanormousemonoclonalantibodiesknowntohaveappropriatekappalightchains;poorforgeneral-purpose(polyclonal)IgGpurification

SupportsOffered

Crosslinked6%beadedagarose

PackageFormats

Resinslurries(2sizes)ChromatographyCartridges(2sizes)PackedSpinColumns(3sizes)NAbIgGPurificationKits(2sizes)


Capacity ProteinL:5-10mghumanIgG/mLresinProteinLPlus:>10-20mghumanIgG/mLresin


Product #

Description

Pkg. Size

U.S. Price

20510 Protein L Agarose 2mL $248

20512 Protein L Agarose 10mL $950

89928 Pierce Chromatography Cartridges, Protein L 2x1mL $190

89929 Pierce Chromatography Cartridge, Protein L 1x5mL $375

89955 NAb Protein L Spin Columns 10x0.2mL $270

89959 NAb Protein L Spin Columns 5x1mL $489

89963 NAb Protein L Spin Column 1x5mL $459

89951 NAb Protein L Spin Purification Kit Kit $365

89981 NAb Protein L Spin Purification Kit Kit $410

21189 Pierce Recombinant Protein L 1mg $121

20520 Protein L Plus Agarose 2mL $365

15190 Protein L, Clear 96-Well Plates 5plates $191

For complete product details,

visit www.thermoscientific.com/pierce or request the Antibody Purification

Handbook (1601974)

Version 2

Thermo Scientific Pierce Antibody Production and Purification Technical Handbook


IgG Binding and Elution Buffers for Protein A, G, A/G and L

Binding and Elution Steps in Affinity PurificationAffinitypurificationproceduresinvolvinginteractionofanantibodywithitsantigengenerallyusebindingbuffersatphysiologicpHandionicstrength.However,manyantibodypurificationmethodsdonotusetheantibody-antigeninteraction;rather,theyinvolvebindingofantibodiesbyimmobilizedligandsthatarenottheantigen.Insuchcases,optimalbindingconditionsaredeterminedbytheuniquepropertiesoftheantibody-ligandinteraction,whichmaybedifferentfromphysiologicpHandionicstrength.

Oncethebindinginteractionoccurs(i.e.,theantibodyis“captured”bytheimmobilizedligand),thesupportiswashedwithadditionalbuffertoremovenonboundcomponentsofthesample.Finally,

elutionbufferisaddedtobreakthebindinginteractionandreleasethetargetmolecule,whichisthencollectedinitspurifiedform.ElutionbuffercandissociatebindingpartnersbyextremesofpH(loworhigh),highsalt(ionicstrength),theuseofdetergentsorchaotropicagentsthatdenatureoneorbothofthemolecules,removalofabindingfactor,orcompetitionwithacounterligand.Inmostcases,subsequentdialysisordesaltingisrequiredtoexchangethepurifiedproteinfromelutionbufferintoamoresuitablebufferforstorageoruse.

ThermoScientificPierceIgGBindingandElutionBuffershavebeenoptimizedtoprovidethehighestpossibleefficiencyofIgGbindingandelutionusingimmobilizedProteinA,ProteinGandProteinA/G.Useofotherbufferformulationsmaysignificantlyalternotonlythebindingcapacitybutalsothevolumesofwashbufferrequiredtoensuregoodpurification.

Binding capacities with different Thermo Scientific Buffers expressed asmg of IgG bound per 2mL of resin.

Immobilized Protein A Immobilized Protein G Immobilized Protein A/G

Serum Sample0.1 M Tris•HCl

pH 8.0Pierce Protein A Binding Buffer

0.1 M Tris•HCl pH 8.0

Pierce Protein G Binding Buffer

0.1 M Tris•HCl pH 8.0

Pierce Protein A Binding Buffer

Rabbit 17.81 33.19 21.51 27.75 13.89 19.61

Sheep 2.15 10.64 25.53 33.33 9.83 15.71

Bovine 6.16 22.76 31.72 48.10 15.13 22.06

Mouse 5.25 7.15 5.65 15.05 4.32 11.49

Rat 4.99 8.30 8.43 11.80 5.20 6.66

Horse 6.25 16.50 36.19 21.46 14.88 17.12

Dog 35.77 22.27 13.38 20.55 21.96 24.60

Chicken 0.91 1.21 1.63 7.27 1.21 4.10

Pig 29.61 24.83 21.25 27.51 19.24 29.48

Human 19.88 25.53 11.68 23.59 9.92 17.67


Description

Highlights

Pkg. Size

U.S. Price

54200 Protein A/G IgG Binding Buffer •EnsuresmaximumrecoveryofIgGfromimmobilizedProteinA/G 240mL $ 37

2100121007

Protein A IgG Binding Buffer •High-yieldisolationofMouseIgG1usingProteinAcolumns•Premixedandeasytouse

1L3.75L

$ 70$195

21019 21011

Protein G IgG Binding Buffer •EnsuresmaximumrecoveryofIgGfromimmobilizedProteinG 1L3.75L

$ 66$194

2100421009

IgG Elution Buffer •High-yieldisolationofIgGfromImmobilizedProteinAandProteinG 1L3.75L

$ 65$165

2102021012

Gentle Ag/Ab Binding Buffer pH 8.0 •Speciallyformulatedandprefiltered•Eliminatesuseofharshacidicelutionconditions

1L3.75L

$103$197

210302102721013

Gentle Ag/Ab Elution Buffer pH 6.6 •Speciallyformulatedforneutralelutions•Notcompatiblewithphosphatebuffers

100mL500mL3.75L

$ 98$ 90$274

21016 IgM Binding Buffer •SpeciallyformulatedforoptimalbindingofmouseIgM 800mL $ 77

21017 IgM Elution Buffer •SpeciallyformulatedforoptimalrecoveryofmouseIgM 500mL $ 63

21018 MBP Column Preparation Buffer •SpeciallyformulatedforusewithImmobilizedMBPandIgMPurificationKit 50mL $ 33

21034 Mouse IgG1 Mild Elution Buffer •SeparateIgG1fromotherIgGsubclasses 500mL $ 80

21033 Mouse IgG1 Mild Binding and Elution Buffer KitIncludes:PierceProteinAIgGBindingBuffer MouseIgG1MildElutionBuffer PierceIgGElutionBuffer

•CompletekittoallowmouseIgG1tobeseparatedfromothermouseIgGsubclasses

Kit1L500mL1L

$255



Thermo Scientific Melon Gel Purification Products

Melon™GelProductsprovideanexcitingnewapproachtopurifyingmonoclonalandpolyclonalantibodiesfromserum,tissueculturesupernatantandascitesfluid.MelonGelworkswithantibodiesfromavarietyofspeciesandsubclasses,manyofwhichdonotpurifyefficientlywithProteinAorProteinG.BecauseMelonGelisnotabind-and-releasesupport,itisextremelyfastandgentletoyourantibodies,resultinginantibodypreparationsofhighpurityandhighactivity!

How does it work? MelonGelcontainsaproprietaryligandthatretainsmostproteinfoundinserum,ascitesandculturesupernatants,whileallowingIgGtopassthroughthesupportandbecollectedintheflow-throughfraction.TheresultingrecoveryandpurityoftheIgGisolatedbythismethodrivalsthatobtainedfromthesamesamplesusingbind-and-releasesupportssuchasProteinAorProteinG.

Highlights:• Simple, one-step protocol–notediousbinding,washing,and

multipleelutionsteps• Rapid purification–purifiesantibodiesfromserumfourtosix

timesfasterthanProteinAorGmethods• High recovery and purity–antibodiesfrommanyspecies

arerecoveredwithgreaterthan90%yieldandgreaterthan80%purity

• Robust purification–workswithawiderangeofantibodiesincludingmanythatdonotpurifywellonProteinAorProteinG

• Gentle purification–noharshelutionconditionsmeansantibod-iesretainmoreactivity

• Reusable support–MelonGelSupportcanbeusedformultipleantibodypurifications

• Available in various formats–spincolumns,purificationkitsandchromatographycartridgesforantibodypurificationfromserum,ascitesandculturesupernatant

IgG purification performance of the Thermo Scientific Melon Gel System, Protein A and Protein G.

Source Melon Gel Protein A Protein G

Human H H H

Mouse H H H

Rabbit H H H

Rat H L M

Goat H L M

Cow M L H

Sheep M L H

Horse H L H

GuineaPig H H L

Pig H H L

Chicken N N N

Hamster H M M

Donkey H M H

H = high recovery, M = medium recovery, L = low recover, N = no recovery


Description

Pkg. Size

U.S. Price

45206 Melon Gel IgG Spin Purification Kit Kit $ 243

45212 Melon Gel IgG Spin Purification Kit Kit $ 507

45214 Melon Gel Monoclonal IgG Purification Kit Kit $1106

45216 Saturated Ammonium Sulfate Solution 1L $ 147

45219 Ascites Conditioning Reagent 5mL $ 35

89932 Pierce Chromatography Cartridges, Melon Gel 2x1 $ 100

89933 Pierce Chromatography Cartridges, Melon Gel 1x5 $ 201

45208 Melon Gel Spin Kit Plate 2plates $ 311

89972 Melon Gel Purification Buffer 1pack $ 13

89973 Melon Gel Regenerant 1pack $ 10

For more data using Melon Gel and for complete

product information, please request the Antibody Purification Technical Handbook (1601974) or visit

www.thermoscientific.com/pierce


Thiophilic Gel Antibody Purification

Thiophilicadsorptionisalow-cost,efficientalternativetoammoniumsulfateprecipitationforimmunoglobulinpurificationfromcrudesamples.Ammoniumsulfateprecipitationmustbefollowedbyseveraladditionalstepstocompletelyremovecontaminantsincrudesamples.Thiophilicadsorptionisasimple,rapid,one-stepmethodforantibodypurificationfromserum,ascitesortissueculturesupernatant.

Thiophilicadsorptionisahighlyselectivetypeoflyotropicsalt-promotedprotein:ligandinteractionphenomenonthathasbeenstudiedextensivelybyPorathandco-workersandotherresearchers.1Thisinteractionistermedthiophilicbecauseitdistinguishesproteinsthatrecognizeasulfonegroupincloseproximitytoathioether.Thiophilicadsorptionincorporatespropertiesofbothhydrophobicandhydrophilicadsorption.However,incontrasttostrictlyhydrophobicsystems,thiophilicadsorptionisnotstronglypromotedbyhighconcentrationsofsodiumchloride.Instead,thiophilicadsorptionispromotedbyincreasedconcentrationsofwater-interacting,non-chaotropicsaltssuchaspotassiumandammoniumsulfate.

ThermoScientificPierceThiophilicAdsorbentis6%beadedagarosemodifiedtocontainsimplesulfone/thioethergroups(seestructureatright).OurThiophilicAdsorbenthasahighbindingcapacity(20mgofimmunoglobulinpermLofresin)andbroadspecificitytowardimmunoglobulinsderivedfromvariousanimalspecies.Notably,thiophilicadsorptionisoneoffewmethodsavailableforpurificationofIgYfromchicken(seealsosubsequentdiscussionofIgYpurification).Amonghumanserumproteins,immunoglobulinsanda2-macroglobulinsarepreferentiallyboundbyourThiophilicAdsorbent.2

PurificationusingPierceThiophilicAdsorbentresultsingoodproteinrecoverywithexcellentpreservationofantibodyactivity.Samplepreparationrequirestheadditionof0.5Mpotassiumsulfatetotheserum,ascitesorculturefluid.GreaterspecificityforimmunoglobulinsisobtainedifthesampleisbufferedatpH8.0.Thegentleelutionconditions(e.g.,50mMsodiumphosphate,pH7-8)yieldconcentrated,essentiallysalt-free,highlypurifiedimmunoglobulinsatnearneutralpH.

Afteruse,ourThiophilicAdsorbentcanberegeneratedbytreatmentwithguanidine•HCl.OurdataindicatethattheAdsorbentcolumncanbeusedatleast10timeswithoutsignificantlossofbindingcapacity.

OS

SOH

OO

Aga

rose

Bea

d

Structure of Thermo Scientific Pierce Thiophilic Adsorbent.

References1.Porath,J.,et al. (1985).FEBS Lett.185,306-310.2.Belew,M.,et al. (1987).J. Immunol. Method102,173-182.3.Hutchens,T.W.andPorath,J.(1987).Biochemistry26,7199-7204.4.Lihme,A.andHeegaard,P.M.H.(1990).Anal. Biochem.192,64-69.5.Unpublishedinternaldocuments.

Thermo Scientific Pierce Thiophilic Adsorbent and Purification KitEconomical purification of mouse antibodies from ascites fluid.

Highlights:•BindstoFabandF(ab´)2fragments•BindstoScFv1•High-capacity(20mg/mL),goodproteinrecoveryandretention

ofantibodyfunction•Broadspecificitytowardimmunoglobulinsderivedfromvarious

animalspecies(seeTable4)•BindschickenIgY(alsoIgG)•Simple,rapid,one-steppurificationformonoclonalantibodies

fromascites;easytoscaleup•Usedtoenrichtheimmunoglobulinfractionfromserumortissue

culturesupernatant•Efficientalternativetoammoniumsulfateprecipitationfor

enrichingantibodiesfromcrudesamples•Gentleelutionconditionsyieldconcentrated,salt-free

immunoglobulinatnearneutralpH•Highdegreeofpurity

Table 4. Binding characteristics of Thermo Scientific Pierce Thiophilic Adsorbent.

Species

Total A280 Bound from 1mL Serum

% Purity by HPLC

Human 4.8 70

Mouse 8.6 63

MouseIgG1 11.6 92

MouseIgG2a 9.3 88

MouseIgG2b 9.8 97

MouseIgG3 10.7 94

Rat 13.0 79

Bovine 17.9 90

Calf 11.1 89

Chicken 5.2 76

Dog 12.2 91

Goat 17.3 92

GuineaPig 11.1 71

Horse 13.0 93

Pig 21.1 90

Rabbit 6.7 84

Sheep 12.3 89 Reference1.Schulze,R.A.,et al.(1994).Anal. Biochem.220,212-214.


Description

Pkg. Size

U.S. Price

20500 Pierce Thiophilic Adsorbent 10mL $ 92

44916 Pierce Thiophilic Purification KitIncludes:ThiophilicAdsorbentColumns BindingBuffer ElutionBuffer ColumnStorageBuffer(2X) Guanidine•HClCrystals ColumnExtenders

Kit4x3mL1,000mL1,000mL100mL230g

$443


IgM Purification

Immobilized Mannan Binding ProteinTodevelopaneffectiveaffinitymatrix,ourscientistsexaminedC1qandanothersimilarlystructuredprotein,mannanbindingprotein(MBP).SerumMBP,likeC1q,iscapableofinitiatingcarbohydrate-mediatedcomplementactivation.MBPisamannoseandN-acetylglucosamine-specificlectinfoundinmammaliansera,andithasconsiderablestructuralhomologytoC1q.8MBPsubunitsareidentical,eachwithmolecularmassofapproximately31kDa(C1qhassixeachofthreedifferentpolypeptidesubunitsofmolecularmass24-28kDa).StudiesinourlabsshowthatMBPdoesnotbindF(ab´)2andFab.

Wehavedevelopedaneasy-to-useThermoScientificPierceImmobilizedMannanBindingProteinandBufferSystemtopurifyIgM.ItismosteffectiveforpurifyingmouseIgMfromascites.PurifiedIgMcanbeobtainedfromasinglepassovertheaffinitycolumn.HumanIgMwillbindtothesupport,albeitwithslightlylowercapacity,andyieldaproductatleast88%pureasassessedbyHPLC.ThepurificationofIgMfromotherspeciesandmouseserumhasnotyetbeenoptimized.

Immobilized MBP and IgM Purification KitEasy IgM purification with guaranteed 88% pure mouse IgM!

0 10 20 30 40 50

0

20

40

60

80

100

Time (min.)

Abs

orba

nce

at 2

80 n

m(p

erce

nt o

f max

.)

IgM

Demonstration of the high purity of MBP-purified IgM from mouse ascites. Theboundmaterialfrommouseasciteswaselutedfromthe5mLMBPcolumnasdescribedintheStandardProtocol.Thehighest280nmabsorbingfractionfromtheelutionwaschromatographedusingtheconditionsdescribedintheinstructions.


Description

Pkg. Size

U.S. Price

22212 Immobilized Mannan Binding ProteinCapacity:~1mgIgM/mLofresin

10mL $936

44897 IgM Purification Kit Kit $610

21016 IgM Binding Buffer 800mL $ 77

21017 IgM Elution Buffer 500mL $ 63

21018 MBP Column Preparation Buffer 50mL $ 33

53123 UltraLink Immobilized Mannan Binding ProteinCapacity:>0.75mgIgM/mLofresin

5mL $493

IgA Purification

Human IgA PurificationJacalinisana-D-galactosebindinglectinextractedfromjack-fruitseeds(Artocarpus integrifolia).Thelectinisaglycoproteinofapproximately40kDacomposedoffouridenticalsubunits.JacalinimmobilizedonsupportssuchasagarosehasbeenusefulforthepurificationofhumanserumorsecretoryIgA1.IgAcanbeseparatedfromhumanIgGandIgMinhumanserumorcolostrum.1IgDisreportedtobindtojacalin.2ImmobilizedjacalinisalsousefulforremovingcontaminatingIgAfromIgGsamples.

BindingofIgAtoimmobilizedjacalinoccursatphysiologicpHandionicstrength,asinphosphatebufferedsaline(PBS).ElutionofboundIgAoccurswithcompetitorligand(e.g.,0.1Mmelibioseor0.1Ma-D-galactose)inPBS.Weofferimmobilizedjacalinoncrosslinked6%agarose.

References1.Roque-Barreira,M.C.andCampos-Neto,A.(1985).J. Immunol. Method134(30),1740-1743.2.Aucouturier,P.,et al.(1987).Mol. Immunol.24(5),503-511.

Immobilized JacalinIdeal for human IgA purification.

Highlights:•IdealforpreparinghumanIgAthatisfreeofcontaminatingIgG•FoundtobindhumanIgA1,butnothumanIgA2–usefulforsepa-

ratingthetwosubclasses

ReferencesKumar,G.S.,et al.(1982).J. Biosci.4,257-261.Roque-Barreira,M.C.andCampos-Neto,A.(1985).J. Immunol.134,1740-1743.Mestecky,J.,et al.(1971).J. Immunol.107,605-607.VanKamp,G.J.(1979).J. Immunol. Method27, 301-305.Kondoh,H.,et al.(1986).J. Immunol. Method88,171-173.


Description

Pkg. Size

U.S. Price

20395 Immobilized JacalinCapacity:1-3mghumanIgA/mLofresinSupport:Crosslinked6%beadedagaroseLoading:4.5mgofjacalin/mLofresin

5mL $201



Biotin

Biotin, also known as vitamin H, is a small molecule (MW 244.3) that is present in tiny amounts in all living cells. The valeric acid side chain of the biotin molecule can be derivatized to incorporate various reactive groups that are used to attach biotin to other molecules.Oncebiotinisattachedtoamolecule,themoleculecanbeaffinity-purifiedusinganimmobilizedversionofanybiotin-bindingprotein.Alternatively,abiotinylatedmoleculecanbeimmobilizedthroughinteractionwithabiotin-bindingprotein,thenusedtoaffinity-purifyothermoleculesthatspecificallyinteractwithit.Weofferbiotin-labeledantibodiesandanumberofotherbiotinylatedmolecules,aswellasabroadselectionofbiotinylationreagentstolabelanyprotein.

O

BiotinMW 244.3

S H

H

Valeric Acid Side Chain

H

H

O

HO

Biotin-Binding Proteins

Avidin–Theextraordinaryaffinityofavidinforbiotinallowsbiotin-containingmoleculesinacomplexmixturetobediscretelyboundwithavidin.Avidinisaglycoproteinfoundintheeggwhiteandtissuesofbirds,reptilesandamphibians.Itcontainsfouridenti-calsubunitshavingacombinedmassof67,000–68,000daltons.Eachsubunitconsistsof128aminoacidsandbindsonemoleculeofbiotin.Theextentofglycosylationonavidinishigh;carbohy-drateaccountsforabout10%ofthetotalmassofthetetramer.Avidinhasabasicisoelectricpoint(pI=10–10.5)andisstableoverawiderangeofpHandtemperature.Extensivechemicalmodificationhaslittleeffectontheactivityofavidin,makingitespeciallyusefulforproteinpurification.However,becauseofitscarbohydratecontentandbasicpI,avidinhasrelativelyhighnonspecificbindingproperties.

Streptavidin–Anotherbiotin-bindingproteinisstreptavidin,whichisisolatedfromStreptomyces avidiniiandhasamassof75,000daltons.Incontrasttoavidin,streptavidinhasnocarbohydrateandhasamildlyacidicpI(5.5).ThermoScientificPierceStreptavidinisarecombinantformhavingamassof53,000daltonsandanear-neutralpI.Streptavidinismuchlesssolubleinwaterthanavidin.Thereareconsiderabledifferencesinthecompositionofavidinandstreptavidin,buttheyareremarkablysimilarinotherrespects.Streptavidinisalsoatetramericprotein,witheachsubunitbindingonemoleculeofbiotinwithaffinitysimilartothatofavidin.Guanidiniumchloridewilldissociateavidinandstreptavidinintosubunits,butstreptavidinismoreresistanttodissociation.StreptavidincontainsanRYDsequencesimilartotheRGDsequencethatbindscellsurfacereceptors.TheRYDsequencecancausebackgroundinsomeapplications.

NeutrAvidin Protein–Wealsoofferadeglycosylatedversionofavidin,knownasNeutrAvidinProtein,withamassofapproximate-ly60,000daltons.Asaresultofcarbohydrateremoval,lectinbind-ingisreducedtoundetectablelevels,yetbiotin-bindingaffinityisretainedbecausethecarbohydrateisnotnecessaryforthisactiv-ity.NeutrAvidinProteinofferstheadvantagesofanear-neutralpI(6.3)tominimizenonspecificadsorption,alongwithlysineresiduesthatremainavailableforderivatizationorconjugation.NeutrAvidinProteinyieldsthelowestnonspecificbindingamongtheknownbiotin-bindingproteinsduetoitsnear-neutralpIandlackofbothcarbohydrateandRYDsequence.

Avidin:Biotin Binding


A Comparison of Biotin-Binding Proteins

Thestrongassociationbetweenavidinandbiotincanbeusedinthefieldofaffinityseparations.Byattachingavidintoasolidsupport,abiotinylatedproductcanbeanchoredtothesamesolidsupport.TheattachmentisstableoverawiderangeofpH,saltconcentrationsandtemperatures.Todissociatebiotinfromavidin,8Mguanidine•HCl,pH1.5orboilinginSDS-PAGEsamplebuffermustbeused.

Avidin

Streptavidin

Thermo Scientific NeutrAvidin Protein

Molecular Weight 67K 53K 60K

Biotin-binding Sites 4 4 4

Isoelectric Point (pl) 10 6.8–7.5 6.3

Specificity Low High Highest

Affinity for Biotin (Kd) 10-15M 10-15M 10-15M

Nonspecific Binding High Low Lowest

Immobilized Avidin Products

Strong biotin interaction creates a nearly irreversible bond.

Immobilizedavidincanbeusedinavarietyofapplicationsfortheaffinitypurificationofbiotinylatedmacromolecules.Inonevariation,anantibodythathasanaffinityforaparticularantigenislabeledwithbiotin.Cellscontainingtheantigenarelysed,thenincubatedwiththebiotinylatedantibodytoformatypicalantigen/antibodycomplex.Toisolatetheantigen,thecrudemixtureispassedthroughanimmobilizedavidinorstreptavidincolumn,whichwillbindthecomplex.Afterappropriatewashes,theantigencanbeelutedfromthecolumnwithalowpHelutionbuffer.Thebiotinylatedantibodyisretainedbythecolumn.

Applications:•Bindingbiotinylatedanti-transferrinforpurifyingtransferrin fromserum1

•BindingbiotinylatedpeptidesandelutionwithanSDS/ureasolution2

•HybridizationofbiotinylatedRNAtoitscomplementaryDNA andbindingtoimmobilizedavidin,withsubsequentelutionofthe single-strandedDNA3

•Purificationofdouble-strandedDNA4

References1.Wilchek,M.andBayer,E.A.(1989).Protein Recognition of Immobilized Ligands.

Hutchins,T.W.,ed.AlanR.Liss,Inc.,pp.83–90.2.Swack,J.A.,et al.(1978).Anal. Biochem.87,114–126.3.Manning,J.,et al.(1977).Biochemistry16, 1364–1370.4.Pellegrini,M.,et al.(1977).Nucleic Acids Res.4,2961–2973.


Description

Pkg. Size

U.S. Price

20219 Avidin Agarose Resin Support:Crosslinked6%beadedagaroseCapacity:≥20μgbiotin/mLresin

5mL $150

20225 Avidin Agarose Resin SupportandCapacity:Sameasabove

5x5mL $533

20362 Avidin Agarose ColumnsSupportandCapacity:Sameasabove

5x1mL $190

Immobilized Streptavidin Products

Same high biotin-binding affinity as avidin with low nonspecific binding.

Applications:•Purificationofmembraneantigensinconjunctionwith biotinylatedmonoclonalantibodies1,2

•Cell-surfacelabelingwithbiotinylationreagents,followedby precipitationwithimmobilizedstreptavidin3

•Purificationofcell-surfaceglycoproteinsusingbiotinylated ConcanavalinA4

•Recoveryofsingle-strandedDNAfordideoxysequencing5

References1.Gretch,D.R.,et al.(1987).Anal. Biochem.163,270–277.2.Updyke,T.V.andNicolson,G.L.(1984).J. Immunol. Method73,83–95.3.Lisanti,M.P.,et al.(1989).J. Cell Biol.109,2117–2127.4.Buckie,J.W.andCook,G.M.(1986).Anal. Biochem.156(2),463–472.5.Baqui,M.,et al.(2003).J. Biol. Chem.278,1206–1211.


Description

Pkg. Size

U.S. Price

20347 Streptavidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:1–3mgbiotinylatedBSA/mLresin 15–28μgbiotin/mLresin

2mL $178

20349 Streptavidin Agarose ResinSupportandCapacity:Sameasabove

5mL $310

20353 Streptavidin Agarose Resin SupportandCapacity:Sameasabove

10mL $440

20351 Streptavidin Agarose ColumnsSupportandCapacity:Sameasabove

5x1mL $318

53113 Streptavidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥2mgbiotinylatedBSA/mLresin ≥24μgbiotin/mLresin

2mL $212

53114 Streptavidin UltraLink ResinSupportandCapacity:Sameasabove

5mL $370

53116 Streptavidin Plus UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥4mgbiotinylatedBSA/mLresin ≥48μgbiotin/mLresin

2mL $285

53117 Streptavidin Plus UltraLink ResinSupportandCapacity:Sameasabove

5mL $410

20357 High Capacity Streptavidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>10mgbiotinylatedBSA/mLofresin

2mL $225

20359 High Capacity Streptavidin Agarose ResinSupportandCapacity:Sameasabove

5mL $350

20361 High Capacity Streptavidin Agarose ResinSupportandCapacity:Sameasabove

10mL $600

21344 MagnaBind Streptavidin BeadsSupport:1–4μm,ironoxideparticlesCapacity:2μgbiotin/mLbeads

5mL $271

88816 Pierce Streptavidin Magnetic Beads 1mL $213

88817 Pierce Streptavidin Magnetic Beads 5mL $721


Immobilized NeutrAvidin Products

Less nonspecific binding produces cleaner results and better yields.

Whennonspecificbindingisaprobleminyourapplication,ThermoScientificImmobilizedNeutrAvidinProductsaresuperioralternativestoavidinorstreptavidin.NeutrAvidinBiotin-BindingProteinisamodifiedavidinderivativethatcombinesseveralkeyfeaturestoprovidebiotin-bindingwithexceptionallylownonspecificbindingproperties.

Highlights:•Carbohydrate-free–justlikestreptavidin,NeutrAvidinBiotin- BindingProteinhasnocarbohydrate,eliminatingnonspecific bindingproblemsduetosugars•Nointeractionwithcellsurfacemolecules–absenceofthe Arg-Tyr-Aspsequence(presentinstreptavidin),whichmimics theuniversalcellsurfacerecognitionsequencepresentina varietyofmolecules,eliminatescross-reactivityofcellsurface molecules•Neutralpl–withaplof6.3,NeutrAvidinProteinhasaplthatis closertoneutralitythanavidinorstreptavidin,eliminating electrostaticinteractionthatcontributestononspecificbinding

Applications:•Immunoprecipitation•Purifyingproteinsthatbindtobiotinylatedligands•Capturingbiotinylatedcell-surfaceproteins1-3

•Purifyingbiotinylatedpeptides4


Description

Pkg. Size

U.S. Price

29200 NeutrAvidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>20μgor80nmolbiotin/mLresin (approx.1–2mgbiotinylatedBSA/mLresin)

5mL $205

29201 NeutrAvidin Agarose Resin SupportandCapacity:Sameasabove

10mL $350

53150 NeutrAvidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:12–20μgbiotin/mLgel

5mL $255

53151 NeutrAvidin Plus UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥30μgbiotin/mLgel

5mL $340

29202 High Capacity NeutrAvidin Agarose ResinSupport:Crosslinked6%beadedagaroseCapacity:>75μgbiotin/mLresin >8mgbiotinylatedBSA/mLresin

5mL $250

29204 High Capacity NeutrAvidin Agarose ResinSupportandCapacity:Sameasabove

10mL $410

Immobilized Monomeric Avidin and Kit

Ideal affinity support for gentle, reversible binding of biotinylated proteins.

Tobreaktheavidin-biotininteraction,8Mguanidine•HClatpH1.5orboilinginSDS-PAGEsamplebufferisrequired.Theseelutionmethodsmayresultindenaturationofthebiotinylatedproteinandcauseirreversibledamagetothesupport.Inaddition,avidinorstreptavidinwillbeirreversiblydenaturedandlosetheabilitytobindsubsequentbiotinylatedsamples.

Whenavidiniscoupledtoasolidsupportasthesubunitmonomer,thespecificityforbiotinisretained,buttheaffinityforbiotinbind-ingsubstantiallydecreases(Ka~108M-1).TheMonomericAvidinAgaroseResinandKitcanbeusedtobindbiotinylatedmolecules,andtheboundmaterialcanbecompetitivelyelutedusing2mmbiotininphosphate-bufferedsaline(PBS).Thistechniqueprovidesthegentlestelutionconditionswithoutcontaminationoftheavidinsubunitsorsubstantiallossofcolumn-bindingcapacity.

Highlights:•Purifiesbiotinylatedproductsundermildelutionconditions•Canberegeneratedandreusedatleast10times•Exhibitslittlenonspecificbinding(3%orless)


Description

Pkg. Size

U.S. Price

20228 Monomeric Avidin Agarose Resin Support:Crosslinked4%beadedagaroseCapacity:≥1.2mgbiotinylatedBSA/mLresin

5mL $355

20267 Monomeric Avidin Agarose ResinSupportandCapacity:Sameasabove

10mL $570

20227 Monomeric Avidin Agarose KitSupportandCapacity:SameasaboveIncludes:1x2mLColumn,BindingandElutionbuffers

Kit $341

53146 Immobilized Monomeric Avidin UltraLink ResinSupport:UltraLinkBiosupportCapacity:≥1.2mgbiotinylatedBSA/mLresin

5mL $399

29129 Biotin 1g $ 54

Thermo Scientific Products for Avidin:Biotin Binding


Immobilized Iminobiotin and Biotin

Iminobiotin offers mild dissociation conditions at pH 4.

O

NHHN

S

NH

Immobilized Iminobiotin

HN

HN

HN

Aga

rose

Bea

d

Iminobiotinistheguanidoanalogofbiotin.Thedissociationconstantoftheavidin-iminobiotincomplexispH-dependent.AtpH9.5-11.0,theavidin-iminobiotincomplexwillbindtightly.AtpH4,theavidin-iminobiotincomplexwilldissociate.Becausedenaturingagentssuchas8Mguanidine•HCIor4Mureaarenotusedinthepurification,anavidinconjugatehasabetterchanceofmaintainingitsactivityduringpurification.

UseimmobilizedD-Biotinasan“irreversiblelinkage”tobindstreptavidinconjugates.Thebiotin-streptavidininteractioncanwithstandextremesinpH,saltanddetergents.


Description

Pkg. Size

U.S. Price

20221 Iminobiotin Agarose ResinSupport:Crosslinked6%beadedagaroseSpacer:DiaminodipropylamineCapacity:≥1mgofavidin/mLresin

5mL $119

20218 Biotin Agarose ResinSupport:PierceCDISupportSpacer:DiaminodipropylamineCapacity:≥2mgofavidin/mLresin

5mL $ 99


Thermo Scientific Pierce Chromatography Cartridges are convenient, reliable, ready-to-use, pre-packed 1mL and 5mL columns of sample-prep and affinity purification resins for manual or automated liquid chromatography (LC).

Thecartridgefittingsarecompatiblewiththepopularautomatedliquid-chromatographysystemsorformanualsyringeprocessing.ThecartridgesattachdirectlytoÄKTAorFPLCSystemswithoutadditionalconnectors.Cartridgescanbeusedindividuallyorconnectedinaseriestoobtainevenhighercolumncapacity.EachproductsuppliedinthePierceChromatographyCartridgeformataccessorypackthatreadilyadaptscartridgesforuseusingLuer-LokSyringeFittingsortubing.Thecartridgesprovidefast,easyandreproduciblechromatographicseparationsandcanberegeneratedformultipleuses.

Thermo Scientific FPLC Cartridges shematic.

Chromatography Cartridge Highlights• Two sizes–1mLand5mL,convenientfortypicalresearchscales• Compatible–fittingsallowconnectionwithpopularLCsystems

orastandardsyringe• Versatile–usesinglyorconnectedinseriestoservicedifferent

capacityrequirements• Validated–availablecartridgeshavebeentestedtoensure

performanceintheformat• Reusable–accessorypackincludescapsforconvenient

storagebetweenuses• Economical–comparableperformanceatlowercostthanother

commerciallyavailablecartridges

Thermo Scientific Pierce Chromatography Cartridge properties.

1mL Cartridge 5mL Cartridge

Dimensions 0.7x2.7cm 1.3x3.8cm

RecommendedFlowRate 1-4mL/min 1-7mL/min

MaximumPressure 0.3mPa(43psior3bar) 0.3mPa(43psior3bar)

CartridgeMaterial polypropylene polypropylene

FritMaterial polyethylene polyethylene

Applications for Pierce Chromatography Cartridges:•His-taggedproteinpurification•GST-taggedproteinpurification•Antibodypurification•Biotinbinding•Phosphoproteinenrichment•Proteindesalting

FPLC Cartridge Overview

20mm

Port Diameter: 4.2mm10-32 threaded coned port

14mm

4mm

17.5

mm

17.5

mm

89m

m 13.5mm

Port Diameter: 4.2mm10-32 threaded coned port

8.5mm

4mm

12.5

mm

75m

m


His-tagged Protein FPLC Purification

Thermo Scientific HisPur Nickel-NTA CartridgesDelivers the highest yield of His-tagged protein with a re-useable resin.

Highlights:•Bindupto60mgof6xHis-taggedproteinpermilliliterofresin•Purifyproteinsusingnativeordenaturingconditions•UsewithPierceCellLysisReagentsandavarietyofbufferadditives•Reusecartridgesseveraltimes

0 20 40 60 80 100 120 140mL0

500

1000

1500

2000

2500

3000

3500

4000

A28

0

M L FT W E

FT W E

Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Nickel-NTA Cartridge. Bacteriallysate(130mgtotalprotein)containingover-expressed6xHis-GFP(greenfluorescentprotein)wasdiluted1:1withequilibra-tionbufferandappliedtoaHisPurNi-NTAChromatographyCartridgeataflowrateof1mL/min.ThecartridgewaswashedwithPBS,68mMimidazoleuntilthebaselineabsorbancewasreached.The6xHis-GFPwaselutedwithPBS,300mMimidazole.Left panel:6xHis-GFPelutionwasmonitoredat280nm(blackline)and485nm(redline;GFP-specific).Right panel:SelectedfractionswereanalyzedbySDS-PAGE.Gellaneswerenormalizedtoequivalentvolume.M =MWmarker,L =lysateload,FT =flow-throughandE =elution.

Seepage14formoreinformationandotherpackagingformatsofHisPurNi-NTAResin.


Description

Pkg. Size

U.S. Price

90098 HisPur Ni-NTA Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding up to 60mg of His-tagged protein per cartridge.

5cartridges $155

90099 HisPur Ni-NTA Chromatography Cartridges, 5mL Formulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding up to 300mg of His-tagged protein per cartridge.

2cartridges $200

Thermo Scientific HisPur Cobalt CartridgesHighly selective binding for the highest purity His-tagged protein purification.

Highlights:•Obtainmorethan10mgofpureHis-taggedproteinpermilliliter

ofresinwithoutoptimizingimidazolewashingconditions•Cobalt-chelatecoordinationcorebindsfewerhostproteincon-

taminants,resultinginlowerbackgroundthannickelresins•Nometalcontaminationinelutedhistidine-taggedproteinsample•Purifyproteinsundernativeordenaturingconditions;compatible

withPierceCellLysisReagentsandavarietyofbufferadditives•Reusecartridgesseveraltimes

0

1000

2000

3000

4000

5000

6000

0 5 10 15 20 25mL

Flow-through Wash Elution

A28

0

Flow-through (FT)M S Wash Elution

Purification of 6xHis-GFP from E. coli lysate using a Thermo Scientific HisPur Cobalt Cartridge. His-taggedgreenfluorescentprotein(GFP)wasextractedfromE. coliusingThermoScientificB-PERBacterialProteinExtractionReagentinPhosphateBuffer(Product#78266)containingThermoScientificHaltProteaseInhibitorCocktail,EDTA-Free(Product#78415).Thelysatewasdiluted1:1withequilibration/washbuffer(50mMsodiumphosphate,300mMsodiumchloride,10mMimidazole,pH7.4)andappliedtoaHisPurCobaltChromatographyCartridgeataflowrateof0.3mL/min.Thecartridgewaswashedwithequilibration/washbufferuntilthebaselineabsorbanceat280nmwasreached.His-taggedGFPwaseluted(50mMsodiumphosphate,300mMsodiumchloride,150mMimidazole;pH7.4)andselectedfractionswereanalyzedbySDS-PAGEandThermoScientificGelCodeBlueStainReagent(Product#24592).M =MWMarker;S=non-fractionatedlysate;FT =flow-through.

Seepage16formoreinformationandotherpackagingformatsofHisPurNi-CobaltResin.



Description

Pkg. Size

U.S. Price

90093 HisPur Cobalt Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 10mg of His-tagged protein per cartridge.

5cartridges $160

90094 HisPur Cobalt Chromatography Cartridges, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 50mg of His-tagged protein per cartridge.

2cartridges $267

GST-Tagged Protein FPLC Purification

Thermo Scientific Pierce Glutathione Agarose CartridgesCost-effective and high-performance GSH resins for purification of recombinant GST fusion proteins.

Highlights:•Bindsatleast25mgofrecombinantGSTproteinpermilliliter

ofresin•Consistentlypurifiesatleast10mgofGST-taggedproteinper

milliliterofresinwithgreaterthan90%purity•Economicallypricedandcanbereusedatseveraltimeswithout

reductioninbindingcapacityandpurificationperformance•WorkswelltopurifyGST-fusionproteinsfrombacteriallysates

orusewithpre-purifiedGST-taggedproteinstopulldownproteininteractions

•ValidatedandeffectiveforusewithPierceCellLysisReagentstoextractandpurifyfrombacterialormammaliancellcultures

Seepage18formoreinformationandotherpackagingformatsofPierceImmobilizedGlutathioneAgaroseResin.

0

1000

2000

A28

0

3000

4000

mAU

SampleApplication

Wash Elutionof

11.8 mg pureGST-fusion protein

0.0 10.0 20.0 30.0 40.0 50.0 60.0mL

Purification of Pak 1-GST fusion protein on Thermo Scientific Pierce Glutathione Chromatography Cartridge 1mL.

Sample:50mg(10mL)clarifiedE. colilysatecontainingexpressedGST-Pak1,M34,000BindingBuffer:5mMTris,150mMsodiumchloride,pH8.0ElutionBuffer:50mMTris,150mMsodiumchloride,10mMGlutathione,pH8.0FlowRate:Loadat0.5mL/min,Wash,Elute1mL/minInstrument:AKTApurifier

16.5

47

3225

11080

210

M Load FT Wash Elution

Purification of Pak 1-GST fusion protein using the Pierce GST Spin Purification Kit (Product # 16106). Pak1-GSTlysate(2.4mgtotalprotein)wasappliedinGlutathioneBindingBuffertoa0.2mLPierceGlutathioneSpinColumnandelutedwith10mMGlutathioneElutionBuffer,pH8.0.FractionswereresolvedbySDS-PAGEusinga4-20%Tris-Glycinegel.GelwasstainedwithGelCodeBlueStainReagent(Product#24590).Pierce3-ColorProteinMolecularWeightMarkerMix(Product#26691)wasused.


Description

Pkg. Size

U.S. Price

16109 Pierce Glutathione Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding approx. 10mg of GST-tagged protein per cartridge.

5cartridges $230

16110 Pierce Glutathione Chromatography Cartridges, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding approx. 50mg of GST-tagged protein per cartridge.

2cartridges $400

FPLC Cartridges


Antibody FPLC Purification

Thermo Scientific Pierce Protein A Agarose CartridgesIdeal for purification of IgG from serum and other fluids.

Highlights:•Bindstoawiderangeofantibodies–especiallygoodfor

purificationofrabbitIgG•LessexpensivethanProteinGagarose

M L FT EFT

FT E

E

A28

0 A

280

M L FT E

50.0mL40.030.020.010.00.0

50.0mL40.030.020.010.00

500

1000

1500

2000

2500

3000

mAU

0.0

0

500

1000

1500

2000

2500

3000

mAU

Comparable antibody yield and purity achieved with Thermo Scientific Pierce Protein A Chromatography Cartridge. Normalhumanserum(60mg)wasappliedinPBStoa1mLThermoScientificPierceProteinACartridge(top)andaHiTrap®Column(bottom)andelutedwith0.1Mglycine,pH2.8,usingaflowrateof1mL/minute.Thearrowdenotesthestartofthelow-pHelution.TheyieldofhumanIgGwas6.85mgand6.88mg,respectively.FractionswereseparatedbySDS-PAGEandthegelswerestainedwithThermoScientificImperialProteinStain(Product#24615).M =MWmarker,L =sampleload,FT =flow-throughandE =elution.

Seepage60formoreinformationandotherpackagingformatsofPierceProteinAAgaroseResin.


Description

Pkg. Size

U.S. Price

89924 Pierce Protein A Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding 35mg human IgG per cartridge.

2cartridges $170

89925 Pierce Protein A Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-Lokfittings.Sufficient for: Binding 175mg human IgG per cartridge.

1cartridge $335

Thermo Scientific Pierce Protein G Agarose CartridgesEspecially suited for purification of monoclonal antibodies from mouse and the broadest spectrum of species and IgG subclasses from human, goat and sheep samples.

Highlights:•Albuminandcellsurfacebindingsitehavebeenremovedfrom

ProteinGtoprovidehigherantibodypurity•BindstoawiderrangeofantibodiesthanProteinAbeads

M L FT ElutionFT

FT E

E

A28

0A

280

M L FT Elution

35.0mL30.020.0 25.010.0 15.05.00.0

35.0mL30.020.0 25.010.0 15.05.00.0

0

500

1000

1500

2000

2500

3000

mAU

0

500

1000

1500

2000

2500

3000

mAU

Comparable antibody yield and purity acheived with Thermo Scientific Pierce Protein G Chromatography Cartridge. Normalhumanserum(60mg)wasappliedinPBStoa1mLThermoScientificPierceProteinGCartridge(top)anda1mLHiTrapColumn(bottom)andelutedwith0.1Mglycine,pH2.8,usingaflowrateof1mL/minute.ThearrowdenotesthestartofthelowpHelution.FractionswereseparatedbySDS-PAGEandthegelswerestainedwithImperial®ProteinStain(Product#24615).M =MWmarker,L =sampleload,FT =flow-throughandElution=elutionfractions.

Seepage60formoreinformationandotherpackagingformatsofPierceProteinGAgaroseResin


Description

Pkg. Size

U.S. Price

89926 Pierce Protein G Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: Binding 11 to 15mg human IgG per cartridge.

2cartridges $190

89927 Pierce Protein G Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 55 to 75mg human IgG per cartridge.

1cartridge $390


Thermo Scientific Pierce Protein A/G Agarose CartridgesGenetically-engineered protein that combines the IgG binding domains of both Protein A and Protein G.

Highlights:•SinglesupportprovidesallbenefitsofProteinAandProteinG•BindsawiderrangeofantibodiesthanProteinAand

ProteinGbeads

0 500

1000

1500

2000

2500

3000

3500

4000mAU

0.0 10.0 20.0 30.0 40.0mLA1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 B11 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4

A)

PureRabbitIgG

B)

M S FT E

RabbitIgG

kDa210

110

80 47

32 25

A28

0

Thermo Scientific Pierce Protein A/G Chromatography Cartridges are effec-tive for affinity purification of immunoglobins from serum. A) 2mLofrabbitserumwasappliedtothecartridgeandtheresultingchromatogramrecorded.B) FractionswereanalyzedbySDS-PAGEona4-20%TrisGlycinegelstainedwithImperialProteinStain(Product#24615).M=Markerproteins,S=Sampleapplied,FT=Flow-throughduringsampleloadandwash,E=ElutedrabbitIgG.ThearrowindicatesthelocationoftheisolatedIgG.

Seepage61formoreinformationandotherpackagingformatsofPierceProteinA/GAgaroseResin.


Description

Pkg. Size

U.S. Price

89930 Pierce Protein A/G Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: Binding 7mg human IgG per cartridge.

2cartridges $200

89931 Pierce Protein A/G Chromatography Cartridges, 5mL Formulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 35mg human IgG per cartridge.

1cartridge $399

Thermo Scientific Pierce Protein L Agarose CartridgesGreat for purifying ScFv or Fab fragments and monoclonal antibodies containing kappa light chains.

Highlights:•Bindskappalightchainsfromawiderangeofspecieswithout

interferingwithantigen-bindingsites†

•BindstoallclassesofIg(e.g.,IgG,IgM,IgA,IgEandIgD)†

•Bindssingle-chainvariablefragments(ScFv)†

•Doesnotbindbovine,goatorsheepIgs•Doesnotbindtobovineantibodies,makingitidealforpurification

ofmouseIgG†fromcellculturesupplementedwithbovineserum† Note: Lambda light chains and some kappa light chains will not bind. Binding will only occur if the appropriate kappa light chains are present.

0 50100150200250300350

mAU

0.0 10.0 20.0 30.0mLA1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A12 B11 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4

M L Flow-through Wash Eluate

SampleApplication

kDa210

80

47

32 25

16.5

IgA

A)

A28

0

B)

IgAheavychain

kappa lightchain

110

Thermo Scientific Pierce Protein L Chromatography Cartridges isolate and purify immunoglobulin classes IgG, IgM, IgA, IgE and IgD via their kappa light chains.A) HumanIgASerum(2mg)wasappliedin100mMsodiumphosphate,150mMsodiumchloride,pH7.2toa1mLPierceProteinLChromatographyCartridgeandpurifiedataflowrateof1mL/minute.Targetwaselutedin0.1Mglycine,pH2.8.B) FractionswereanalyzedbySDS-PAGEona4-20%Tris-GlycinegelstainedwithGelCodeBlueStainReagent(Product#24590).

Seepage61formoreinformationandotherpackagingformatsofPierceProteinLAgaroseResin.


Description

Pkg. Size

U.S. Price

89928 Pierce Protein L Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding 5 to 10mg human IgG per cartridge.

2cartridges $190

89929 Pierce Protein L Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: Binding 25 to 50mg human IgG per cartridge.

1cartridge $375

FPLC Cartridges


Thermo Scientific Melon Gel IgG Purification ResinsEasily purify IgG from serum in 15 minutes.

Highlights:•Notediousbinding,washingandmultipleelutionsteps•Purifiesantibodiesfromserumfourtosixtimesfasterthan

ProteinAorGmethods•Recoverantibodiesfrommanyspecieswith>80%purity•Noharshelutionconditionsmeansantibodiesretainmoreactivity•Reusablesupport

M L

10.00.0

0

1000

2000

3000

4000

mAU

20.0mL

1 2

FT

R

A28

0

FT R

3 4 1 2

Effective reverse-affinity purfication of antibody with Thermo Scientific Melon Gel Cartridges. Normalhumanserum(1mL)wasappliedtoa1mLMelonGelCartridgeataflowrateof1mL/minute.Proteincontaminantsbindtotheresinwhiletheantibodiesflowthrough(FT).Volumeofrecoveredantibody=4to5mL.Regeneration(R)solutionstripstheboundproteincontaminantssothatthecartridgecanbereusedmultipletimes.ThepurityoftheisolatedantibodywasevaluatedbySDS-PAGE(rightpanel)andstainedwithThermoScientificGelCodeBlueStainReagent(Product#24590).M =MWMarker,L =sampleloaded,FT =flow-throughandR =regenerationfractions.

Seepage63formoreinformationandotherpackagingformatsofMelonGelResin.


Description

Pkg. Size

U.S. Price

89932 Melon Gel Chromatography Cartridges, 1mL Formulation:1mLresincartridgeswithLCandLuer-LokFittings.Sufficient for: IgG purification from 1 to 2mL serum per cartridge.

2cartridges $100

89933 Melon Gel Chromatography Cartridges, 5mL Formulation:5mLresincartridgewithLCandLuer-LokFittings.Sufficient for: IgG purification from 5 to 10mL serum per cartridge.

1cartridge $201

Phosphoprotein FPLC Purification

Thermo Scientific Pierce Phosphoprotein Enrichment KitPurify and isolate phosphoproteins for analysis by Western blotting or mass spectrometry.

Highlights:•Lownonspecificproteincontaminationfromcomplexbiological

samples,suchascellculturelysateandmousetissueextract•Easyspinformatenablesenrichmentofphosphorylatedproteins

inlessthan2hours•Achieveshigheryieldsthanothercommerciallyavailablekits

Phosho-p42/44 MAPKCytochrome-C (12KD)

Non-phosphorylated protein

25ug 10ug FT El25ug 10ug FT El TCL TCL

A)

B)

0

200

400

600

800

1000

1200

mAU

10.00.0 20.0 30.0 40.0 50.0 60.0 70.0mL

Non-phosphorylatedProteinsFlow-Through

ConcentratedPhosphorylatedProteins In Eluate

A28

0

Figure A. Enrichment of phosphorylated proteins from K562 cell lysate, 4mg (10mL), processed using a Thermo Scientific Pierce Phosphoprotein Enrichment Chromatography Cartridge, 1mL.Binding/WashBuffer:50mMMES2-(N-morpholino)ethanesulfonicacid,monohydrate),250mMsodiumchloride,25mMadipicacid,0.25%CHAPS;pH5.0;ElutionBuffer:100mMsodiumphosphate,500mMsodiumchloride,0.25%CHAPS;pH7.5;Flowrateduringsampleapplication0.3mL/min;1mL/minduringwashandelution.Figure B. Thermo Scientific Pierce Phosphoprotein Enrichment Chromatography Cartridge, 1mL, provides high specificity for the enrichment of phosphoproteins from complex biological samples. ConcentratedFlow-throughandElutionfractionswereresolvedonSDS-PAGE.Gellaneswerenormalizedbyproteinconcentration,10mg/lane.Westernblotanalysiswasperformedusingphospho-specificantibodies.CytochromeCisanegativecontrolfornonspecificbindingofnon-phosphorylatedproteins.Highspecific-ityofbindingaffinityforthephosphorylatedtargetwasdemonstratedbythepresenceofPhospho-p42/44MAPKintheEluateandabsence,thereof,intheFlow-through.

Seepage53formoreinformationandotherpackagingformatsofPierceProteinPhosphoproteinEnrichmentResin.


Description

Pkg. Size

U.S. Price

87743 Pierce Phosphoprotein Enrichment Chromatography Cartridges, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Purifying samples containing 4mg total protein (400µg phosphoprotein) per cartridge.

2cartridges $124

87744 Pierce Phosphoprotein Enrichment Chromatography Cartridges, 5mLFormulation:5mLresincartridgewithLCandLuer-Lokfittings.Sufficient for: Purifying samples containing 20mg total protein (2mg phosphoprotein) per cartridge.

1cartridge $310


Biotinylated Protein FPLC Purification

Thermo Scientific Streptavidin Agarose and Neutravidin Agarose CartridgesResins with high biotin affinity and low nonspecific binding.

Highlights:•ExhibithighbindingaffinitytowardsthevitaminBiotin(vitaminH)•Capturesbiotinylatedproteins•Derivativesoftheproteinavidinfromchickeneggwhitesthat

differbytheirsourceandglycosylationlevel

Pierce High Capacity Streptavidin Chromatography Cartridge

0

500

1000

1500

2000

2500

3000

3500

mAU

0.0 20.0 40.0 60.0 80.0mL

Yield655 µgTarget

GE HiTrap Streptavidin HP

0

500

1000

1500

2000

2500

3000

mAU

0.0 20.0 40.0 60.0 80.0mL

Yield469 µgTarget

Pierce High Capacity NeutrAvidin Chromatography Cartridge

0

500

1000

1500

2000

2500

3000

mAU

0.0 20.0 40.0 60.0 80.0mL

Yield388 µgTarget

Thermo Scientific High Capacity Streptavidin Chromatography Cartridge achieves comparable yield and performance in binding of small biotinylated molecule, Biotin 4 Nitrophenyl ester (BpNPE), as compared to HiTrap.

Sample:4.8mgBpNPE(in24mL)ColumnSize:1mLBindingBuffer:0.5MSodiumAcetate,pH5.0ElutionBuffer:0.5MNaOHStreptavidincartridges;0.1MNaOHNeutrAvidincartridgeFlowRate:SampleApplication0.3mL/minute;WashandElution1mL/minuteMonitored2wavelengths:Flowthrough/WashinNaAcetateA270nm(red);ElutioninNaOHA410nm(pink)Instrument:AKTApurifierYielddeterminedbyODA410nm(ExtinctionCoefficientforBpNPE18.3A410nm)

SupplierCartridge

SizeBiotinylated BSA Bound

Pierce High Capacity Streptavidin Chromatography Cartridge

1mL 12.9mg

5mL 75.9mg

GE HiTrap Streptavidin HP

1mL 10.7mg

5mL (Notofferedin5mLsize)

Pierce High Capacity NeutrAvidin Chromatography Cartridge

1mL 12.8mg

5mL 70.mg

Binding capacity of Thermo Scientific High Capacity Streptavidin Chromatography Cartridges is comparable to that of HiTrap. ColumnswereoverloadedwithBiotinylatedBSAandpurifiedpermanufacturer'sinstructions.BindingcapacitywasdeterminedusingtheThermoScientificPierceBCAProteinAssayKit(Product#23225).Note: Capacity for the avidin resins was determined indirectly by subtracting the unbound biotinylated BSA present in the flow-through fractions from the total amount applied to the column.

Comparison of biotin-binding proteins.

Biotin-Binding Protein

Avidin Streptavidin NeutrAvidin

Molecular Weight 67K 53K 60K

Biotin-binding Sites 4 4 4

Isoelectric Point (pI) 10 6.8-7.5 6.3

Specificity Low High Highest

Affinity for Biotin (Kd) 10-15M 10-15M 10-15M

Nonspecific Binding High Low Lowest

Seepages67-68formoreinformationandotherpackagingformatsofSteptavidinandNeutravidinAgaroseResin


Description

Pkg. Size

U.S. Price

87739 High Capacity Streptavidin Chromatography Cartridges, 1mL Formulation:1mLcrosslinked6%beadedagarosecartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 100µg biotin per cartridge (> 10mg biotinylated BSA per cartridge).

2cartridges $176

87740 High Capacity Streptavidin Chromatography Cartridge, 5mL Formulation:5mLcrosslinked6%beadedagarosecartridgewithLCandLuer-Lokfittings.Sufficient for: Binding > 500µg biotin per cartridge (> 50mg biotinylated BSA per cartridge).

1cartridge $240

87741 High Capacity NeutrAvidin Chromatography Cartridges, 1mL Formulation:1mLcrosslinked6%beadedagarosecartridgeswithLCandLuer-Lokfittings.Sufficient for: Binding > 75µg biotin per cartridge (> 8mg biotinylated BSA per cartridge).

2cartridges $149

87742 High Capacity NeutrAvidin Chromatography Cartridge, 5mL Formulation:5mLcrosslinked6%beadedagarosecartridgewithLCandLuer-Lokfittings.Sufficient for: Binding > 375µg biotin per cartridge (> 40mg biotinylated BSA per cartridge).

1cartridge $206

FPLC Cartridges


Protein Desalting

Thermo Scientific Zeba Desalting Chromatography CartridgesThe best protein desalting resin in cartridge format.

Highlights:•Exceptionaldesaltingandprotein-recoverycharacteristics

comparedtoothercommerciallyavailableresins•Successfulwithverydilute(25μg/mL)proteinsamples•Greaterthan95%retention(removal)ofsaltsandother

smallmoleculesandgoodrecoveryofproteinsandothermacromolecules

A28

0 (blu

e)Co

nduc

tivity

(bro

wn)

A28

0 (blu

e)Co

nduc

tivity

(bro

wn)

A28

0 (blu

e)Co

nduc

tivity

(bro

wn)

Thermo Scientific Pierce Chromatography Cartridge

Supplier G Chromatography Cartridge

Supplier B Chromatography Cartridge

mAU

600

500

400

300

200

100

0

0.0 2.0 4.0 6.0 8.0 10.0mL

0.0 2.0 4.0 6.0 8.0 10.0mL

0.0 2.0 4.0 6.0 8.0 10.0mL

0

100

200

300

400

500

600

700mAU

mAU

600

500

400

300

200

100

0

Efficient salt removal and protein recovery with desalting chromatography cartridge. Bovineserumalbumin(1mg)in1MNaClwasappliedto5mLThermoScientificZebaDesaltingCartridge(top)ataflowrateof5mL/minute.CartridgeprofileshowsisocraticelutionofBSA(blue)andNaCldetectedbyconductivity(brown).Greaterthan95%oftheBSAwasrecoveredandmorethan95%ofthewassaltremoved.Resultswerecomparabletothoseobtainedwithdesalt-ingcartridgesfromothersuppliers(resultsfortheThermoScientificCartridgewereessentiallyidenticaltothatobtainedwithmoreexpensiveGEHealthcareandBio-RadProducts(suppliersGandB,respectively).


Description

Pkg. Size

U.S. Price

89934 Zeba Desalting Chromatography Cartridges, 7K MWCO, 1mLFormulation:1mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Desalting 50 to 250µL samples per use.

5cartridges $147

89935 Zeba Desalting Chromatography Cartridges, 7K MWCO, 5mLFormulation:5mLresincartridgeswithLCandLuer-Lokfittings.Sufficient for: Desalting 100 to 1500µL samples per use.

5cartridges $168


In addition to the few affinity supports whose ligands have broad application to many different protein methods, there are many others whose applications are more narrowly defined or are incorporated into kits for very specific purposes. TheseincludekitstoisolatecellsurfaceproteinsusingbiotinylationandNeutrAvidinAgarose,kitstoenrichforphosphoproteins,glyco-proteinsandubiquitinatedproteins.Thestand-aloneresinsonthispageandthenextincludethosethatcanbeusedtoremovetrypsinfromproteindigest,isolatebloodproteins,purifyC-reactiveproteinandcaptureribonucleosides.Inaddition,page80presentsourcompletelineofmagneticbeads,togetherwiththeirmagnetaccessories.

Immobilized Soybean Trypsin Inhibitor

For effective removal of trypsin, chymotrypsin and elastase from protein digests.

Applications:•Purifyingtrypsin,chymotrypsinandelastase1,2

•Removingproteasesfromactivatedpancreaticjuices3

References1.Feinstein,G.,et al.(1974).Euro. J. Biochem.43(3),569–581.2.Peterson,L.M.,et al.(1976).Biochemistry15(12),2501–2508.3.Reeck,G.R.,et al.(1971).Biochemistry10(25),4690–4698.


Description

Pkg. Size

U.S. Price

20235 Immobilized Soybean Trypsin InhibitorSupport: 4%beadedagaroseCapacity:≥6mgtrypsin/mLofresin

2mL $98

Immobilized Pepstatin

An excellent cathepsin binding matrix.

HN

HN

HN

Pepstatin =

R = (4-amino-3-hydroxy-6-methyl) heptanoic acid

Thermo Scientific Immobilized Pepstatin

i-ValValValRAlaR

Pepstatin

Aga

rose

Bea

d

Immobilized Heparin

ReferenceHelseth,Jr.,D.L.andVeis,A.(1984).Proc. Natl. Acad. Sci. USA81,3302–3306.


Description

Pkg. Size

U.S. Price

20215 Immobilized PepstatinSupport:Crosslinked6%beadedagaroseSpacer:DiaminodipropylamineCapacity:1–2mgofpepsin/mLofresin

5mL $159

Additional Thermo Scientific Affinity Supports and Kits


Immobilized Heparin

Use to isolate many blood proteins that have enzymatic activities.

Applications:•Enrichlysatesfornucleicacid-bindingproteins•Isolatemanybloodproteins

Aga

rose

Bea

d

O

OSO3-

OHCOO-

O

NHOSO3-

OH

CH2OSO3-

OO

n

O

Thermo Scientific Immobilized Heparin

ReferenceSmith,P.K.,et al.(1980).Anal. Biochem.109,466–473.


Description

Pkg. Size

U.S. Price

20207 Immobilized HeparinSupport:4%beadedagaroseLoading:≥0.2mgofheparin/mLofresin(determinedbythecolorimetricmethod)

Kit $73

Immobilized p-Aminophenyl Phosphoryl Choline

For C-reactive protein binding.

O

ON+P

O

O

O-

HN

Thermo Scientific Immobilized p-Aminophenyl Phosphoryl Choline

Aga

rose

Bea

d

ReferenceRobey,F.A.andLiu,T.Y.(1981).J. Biol. Chem.256,969–975.


Description

Pkg. Size

U.S. Price

20307 Immobilized p-Aminophenyl Phosphoryl CholineSupport:Crosslinked6%beadedagaroseCapacity:≥3mgofhumanC-reactiveprotein/mLofresin

5mL $202

Immobilized D-Galactose

For lectin and galactosidase binding.

Applications:•Humanalpha-galactosidaseApurification1

•E. coliheatlabileenterotoxinpurification2

•C-typelectinpurification3

•Choleratoxin(CT)purification4,5

O

S

O

O

O OH

OHOHO

OH

Thermo Scientific Immobilized Thio-alpha-D-Galactose

Aga

rose

Bea

d

References1.Yasuda,K.,et al.(2004).Protein Expression and Purification.37,499–506.2.Okamoto,K.,et al.(1998).J. Bacteriol.180,1368–1374.3.Matsumoto,J.,et al.(2001).Development. 128,3339–3347.4.Bowman,C.C.andClements,J.D.,et al.(2001).Infec. Immun. 69,1528–1535.5.Tinker,J.K.,et al.(2003).Infec. Immun. 71,4093–4101.


Description

Pkg. Size

U.S. Price

20372 Immobilized D-GalactoseSupport:6%beadedagaroseCapacity:≥20mgjacalin/mLresin

5mL $145

Immobilized Boronic Acid

For ribonucleoside isolation.

B

OH

OHHN

Thermo Scientific Immobilized Boronic Acid

OOH

Resi

nB

ead

ReferencesVlassara,H.,et al.(1981).Proc. Natl. Acad. Sci. USA78,5190–5192.Gehrke,C.W.,et al.(1978).J. Chromatogr.150,455–476.


Description

Pkg. Size

U.S. Price

20244 Immobilized Boronic AcidSupport:PolyacrylamideresinbeadsCapacity:≥99%bindingandrecoveryof110μmol AMP/mLofresinSpacer:m-aminophenylgroupLoading:100μmolboronate/mLofresin

10mL $190


Thermo Scientific MagnaBind Beads and Supports

A convenient method for isolating biomolecules using affinity binding, while retaining biological activity.

Magneticbeadsareaconvenientaffinitysupportforavarietyofassays,whichalloweasypurificationofthetargetwithoutcolumnsorcentrifugation.Afterabindingstepinanaffinitypurificationprocedure,themagneticparticlesareeasilyandrapidlycollectedbyplacingthemicrocentrifugetubeorreactionvesselnextanappropriaterare-earthmagnet(Figure1).ThermoScientificMagnaBindbeadsrespondrapidlytoMagnaBindMagnetsbutcanbeeasilydispersedandregatheredmultipletimes(i.e.,theywillnotirreversiblyaggregate)becausetheydonothaveanymagneticmemory.MagnaBindBeadsareavailablepre-coatedwithProteinA,ProteinG,streptavidin,anti-mouseoranti-rabbitantibodies.Activatedbeads,witheitheramineorcarboxylgroups,arealsoavailableforattachingotherproteinsoraffinityligandstoamagneticparticle.

Highlights:•Availablepre-coatedwithpopularaffinityligandsorderivatized forcovalentattachmentofproteinsandotherspecificligands•Beadsdonotirreversiblyaggregatebecausetheyhaveno magneticmemory;collectanddispersethebeadsmultipletimes ifneeded•Mostseparationsrequireashortfive-to10-minutebench-top procedure

Applications:•Cellsortingusingpositiveornegativeselection•Proteinpurificationorimmunoassaysusingdirectorindirectmethods

Characteristics of underivatized Thermo Scientific MagnaBind Beads.

Composition Silanizedironoxide

Magnetization 25–35EMU/g

Type of Magnetization Superparamagnetic(nomagneticmemory)

Surface Area >100m2/g

Settling Rate 4%in30minutes

Effective Density 2.5g/mL

Number of Beads 1x108beads/mg

pH Stability Aqueoussolution,abovepH4.0

Concentration 5mg/mL

Note: To establish a microbe-free preparation, MagnaBind Beads can be washed with antibiotic medium or g-irradiated.

Figure 1. Thermo Scientific MagnaBind Magnet for 1.5mL Microcentrifuge Tube. ThermoScientificMagnaBindBeadsinsolutionswithinamicrocentrifugetubearerapidly“pelleted”whenthetubeisplacedinthemagnetizedholder.Magnetsforsixmicrocentrifugetubesand96-wellmicroplatesarealsoavailable.

ReferencesChaudhuri,T.K.,et al.(2001).Cell107,235–246.Newey,S.E.,et al.(2001).J. Biol. Chem.276,6645–6655.Xu,X.,et al.(2001).J. Biol. Chem.276,43221–43230.


Description

Pkg. Size

U.S. Price

21344 MagnaBind Streptavidin Beads 5mL $271

21348 MagnaBind Protein A Beads 5mL $325

21349 MagnaBind Protein G Beads 5mL $325

21354 MagnaBind Goat Anti-Mouse IgG Beads 50mL $256

21356 MagnaBind Goat Anti-Rabbit IgG Beads 50mL $256

21353 MagnaBind Carboxyl Derivatized Beads 5mL $127

21352 MagnaBind Amine Derivatized Beads 5mL $130

21358 MagnaBind Magnet for 96-Well Plate Separator

1magnet $392

21357 MagnaBind Magnet for 1.5mL Microcentrifuge Tube

1magnet $152

21359 MagnaBind Magnet for 6 x 1.5mL Microcentrifuge Tubes

1magnet $305

Thermo Scientific Affinity Supports


ÄKTA, ÄKTApurifier™, HiTrap® and pGEX® are trademarks of GE Healthcare.Phos-Select™ is a tradmark of Sigma-Aldrich Co.NanoLC™ is a trademark of Eskigent.Trisacryl® is a trademark of Pall Corporation.Luer-Lok™ is a trademark of Becton, Dickinson and Company.Brij® is a registered trademark of ICI Americas.Cibacron® is a registered trademark of Ciba Specialty Chemicals, Inc.Triton® is a registered trademark of Rohm & Haas Company.Tween® is a trademark of ICI Americas.

Contact Information

Belgium and Europe, the Middle East and Africa Distributors Tel: +32 53 85 71 84

France Tel: 0 800 50 82 15

The Netherlands Tel: 076 50 31 880

Germany Tel: 0228 9125650

United Kingdom Tel: 0800 252 185

Switzerland Tel: 0800 56 31 40

Email: [email protected] www.thermoscientific.com/perbio

United States Tel: 815-968-0747 or 800-874-3723 Customer Assistance E-mail: [email protected] www.thermoscientific.com

© 2010 Thermo Fisher Scientific Inc. All rights reserved. These products are supplied for laboratory or manufacturing applications only. Unless indicated otherwise on the inside back cover, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. 16

0201

5 1

1/10

Prin

ted

in th

e U.

S.

thermo scientific pierce protein purification …...thermo scientific pierce protein purification...

Documents