1. separasi dan purifikasi
TRANSCRIPT
![Page 1: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/1.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 1/78
Separasi dan
Purifkasi dalam Bioteknologi
Jumeri M. Wikarta,Ph.D
![Page 2: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/2.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 2/78
Fermentation broth
• Combination of insoluble, gelatinous biomass,the nutrient uid, and the soluble metabolitesresulting from the fermentation operation.
• Fluidity for stirring or aeration is ± 3-7% dryweight of biomass.
• Baterial fermentation for !C" will produe abroth of 3% #$ in whih the slurry is &%'by (olume) wet biomass and *&%
interpartile uid.
![Page 3: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/3.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 3/78
Typial Produt !onentrations"ea#ing Fermenters
![Page 4: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/4.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 4/78
![Page 5: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/5.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 5/78
• +f the desired produt is etraellular, itis only neessary to lter the biomassfrom the broth and isolate the produt
from the uid.• +f a produt is intraellular, a ell
disruption step must rst be employed.
• +f the produt is also water soluble, this
disruption should be performed while thebiomass is still in a slurry form.
![Page 6: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/6.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 6/78
/C0$/1 2+40"/54+0!
• Filtration and entrifugation are unitoperations in whih the suspended solids areseparated from the uid phase.
• 6rying is the remo(al of moisture or sol(entfrom solid partiles
• /(aporation is the remo(al of moisture orsol(ent from a solution.
• +n rystalliation, the onditions of a solutionare ad8usted to hange the solubility of oneof the dissol(ed ompounds so that it lea(esthe solution as a solid.
![Page 7: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/7.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 7/78
Separation and $eo#eryTehni%ues
![Page 8: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/8.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 8/78
9 5 : &,9nm
• ;iroltration, ultraltration, and re(erseosmosis are membrane proesses in whihseparation is based on di<erenes in abilityto ow through a thin barrier that separatestwo uids.
• ;iroltration is a hydraulially dri(enproess using a membrane with a pore siein the 9&& to 3&&& 5 range.
• 2ltraltration, the pore sie is 9&-9=> 5,while for re(erse osmosis, the pore sie is3-9& 5.
![Page 9: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/9.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 9/78
• 5dsorption, ion ehange, olumn hromatography,and a?nity hromatography grouped as reo(erytehni@ues in whih the remo(ed ompound or
solute establishes an e@uilibrium between sites ona solid phase material and the solution.
• +n adsorption, the remo(ed speies is bonded tothe solid phase material by polarity or weaA
hemial bonds.• +on ehange reo(ers material by the interhangeof ions between the li@uid and solid phases.
• Column hromatography may use adsorpti(e, ionehange or moleular sie(e materials to separate
solutes whih are rst loaded onto a olumn ofthe separation material and then eluted in suh amanner that the indi(idual solutes are olleted inseparate frations.
• +n a?nity hromatography, the remo(ed speies isbound with a high le(el of seleti(ity to ligands
![Page 10: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/10.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 10/78
• +n sol(ent etration, the remo(ed ompoundestablishes an e@uilibrium distribution betweenimmisible sol(ents, usually water and an organili@uid.
• /letrophoresis and eletrodialysis are separationtehni@ues that separate harged moleules orions using an eletri eld.
• /letrophoresis separates harged omponents byaentuating small di<erenes in ioni mobility inan eletri eld using a mo(ing arrier uid.
• /letrodialysis onentrates omponents on thebasis of eletromigration through ioni membranes.
![Page 11: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/11.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 11/78
Per&orated bo'l or basket typeentri&uge
•2sed with a lterbag of nylon,terylene, or otton.
• 4he feed is added
ontinually.•#hen the bowl islled with thebiomass aAe, freshwashing li@uid an
be added todisplae the residualbroth uid retained
in the aAe.
![Page 12: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/12.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 12/78
Tubular bo'l entri&uge
5 ontinuousfeed of materialpasses through themahine. 5s soon
as the bowl is fullof the biomassaAe, theentrifuge isstopped, strippeddown, the aAeremo(ed, themahine leanedand
the se@uene
![Page 13: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/13.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 13/78
Multi hamber solid bo'l entri&uge
•5llows three or moretime the apaity ofthe tubular bowl.
• 4he disassembly and
leaning of thisentrifuge is moredi?ult.
![Page 14: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/14.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 14/78
(o))le disk entri&uge
• 0perate ontinuouslysine the solids areautomatially remo(edfrom the bowl.
• 6isharges a ontinuousstream of onentratedsolid-slurry.
![Page 15: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/15.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 15/78
Desludger or intermittent dishargeentri&uge
as anintermittentdisharge ofsolids with lower
water ontentthan the noleentrifuge.
![Page 16: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/16.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 16/78
Sroll entri&uges
2ses a srew within a rotatingbowl to allow ontinuous remo(al
of the solid and the li@uidortions.
![Page 17: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/17.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 17/78
!harateristis o& Di*erent Types o&!entri&uges
![Page 18: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/18.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 18/78
$eo#ery andpurifation o&itri aid
![Page 19: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/19.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 19/78
+solationproedure &or
sodiumla#ulanate
![Page 20: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/20.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 20/78
Purifationproedure &ormirooalnulease
![Page 21: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/21.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 21/78
eo(ery proessesfor human insulin,humangrowth hormone andhuman leuAoyteinterferon synthesiedfrom /. oli
![Page 22: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/22.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 22/78
Why puri&y Proteins
• Charaterie – Funtion
– 5ti(ity
– !truture
• !tudy protein regulation and proteininterations
• 2se in assays
• "rodue 5ntibodies• "erform strutural analysis by -ay
and Crystallography
![Page 23: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/23.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 23/78
-o' to puri&y Proteins
Biohemists eploit the ways indi(idualproteins di<er from one another, suh assolubility, sie, or harge.
!pei
binding
![Page 24: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/24.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 24/78
!lassial Protein Purifation MethodsThe ldest/ 0mmonium Sul&ate
Frationation
![Page 25: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/25.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 25/78
Dialysis 1 2etting $id o& 3(-456S4 or
!hange Bu*ers
![Page 26: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/26.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 26/78
+solation of "roteins fromCells;any di<erent proteins eists within one ell
D ;any steps needed to etrat protein ofinterest, and separate from many
ontaminants
D Before puriation begins, protein mustbe released from ell by
homogeni)ation
![Page 27: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/27.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 27/78
Compartmentalization provides an opportunity
for a purification step
e
Protein profile for compartments of gram-negative prokaryotes
![Page 28: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/28.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 28/78
!ell Disruption
!hemial/ alAali, organi sol(ents,detergents
7n)ymati/ lysoyme, gluanases,hitinase
Physial/ osmoti shoA,freeeEthaw
Mehanial/ soniation,homogeniation, wetmilling, Frenh press
![Page 29: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/29.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 29/78
!hemial Disruption
Detergents suhas Trition 89:;; or(P4; anpermeabili)e ellsby solubili)ingmembranes.
Detergents an bee<pensi#e,denatureproteins, andmust be remo#ed
![Page 30: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/30.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 30/78
Press
!ells are plaedin a stainless
steel ontainer.0 tight fttingpiston is
inserted andhigh pressuresare applied to&ore ellsthrou h a small
![Page 31: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/31.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 31/78
-omogeni)ation
•!ells are plaed in alosed #essel 3usuallyglass5.
•0 tight ftting plunger isinserted and rotated'ith a do'n'ard &ore.
•!ells are disrupted asthey pass bet'een theplunger and #essel 'all.
•0lso, shaking 'ith glass
beads 'orks, B=T/ Frition > -eat
![Page 32: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/32.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 32/78
Soniation
A sonicator can be
immersed directly into
a cell suspension. Thesonicator is vibrated
and high frequency
sound waves disruptcells.
![Page 33: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/33.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 33/78
6i<erential entrifugation
![Page 34: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/34.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 34/78
Protein purifation
Protein mixture applied to column
Solvent (buffer) applied to top,
flowed through column
Different proteins interact with
matrix to different extents, flow
at different rates
Proteins collected separately indifferent fractions
![Page 35: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/35.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 35/78
Column Chromatography
9.!+/GHel ltration '!ieelusion )
=.C5H/G+on ehange
3.!"/C+F+C B+6+HG5?nity
Moleules an be separated on the basis o&/
![Page 36: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/36.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 36/78
ow #e Het "roteins 0ut ofCells
![Page 37: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/37.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 37/78
$eyling Preparati#e-P"!
![Page 38: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/38.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 38/78
$eyling Preparati#e-P"!
![Page 39: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/39.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 39/78
Hel Filtration
!eparates moleules aording to di<erenes in
sie as they pass through a gel ltration mediumpaA in a olumn
5 Aey position in the puriation of thousandsenymes, polyssaharides, nulei aids, protein,
and other biologial maromoleules.;oleules do not bind to the hromatographymedium so bu<er omposition does not diretlya<et resolution
4he li@uid inside the pores is sometimes re<eredto as the stationary phase and this li@uid is ine@uilibrium with the li@uid outside the partiles,re<ered to as the mobile phase
![Page 40: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/40.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 40/78
Hel Filtration
Can be performed in the presene of essential
ions or ofator, detergent, urea, at high or lowioni strength, at 37IC or in the old roomaording to the re@uirements of the eperiment
4he sample one mo(es down the bed as eluent
is added to the top 4he small moleules whih di<use into the gelbeads are delayed in their passage down theolumn ompare with the large moleules whih
anJt di<use into the gel and mo(e ontinuouslydown the olumn in the owing eluent
4he large moleules thus lea(e the olumn rstfollowed by the smaller moleules in the order intheir sie
l fltration hromatography
![Page 41: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/41.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 41/78
l fltration hromatography
![Page 42: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/42.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 42/78
!ie /lusionEHel-ltration
![Page 43: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/43.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 43/78
Si)e9e<lusion hromatography
!ie elusion
![Page 44: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/44.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 44/78
!ie-elusionhromatography
5bsorbane at =K& is used to identifyprotein-ontaining frations. 1ou analso perform an enyme spei assay.
![Page 45: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/45.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 45/78
!olumns !onneted toFration !olletors
![Page 46: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/46.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 46/78
/lution prole 'hromatogram) of gel ltration
![Page 47: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/47.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 47/78
$esolution in gel fltration
Fators in?uene /- !ample (olume- 4he ratio of sample(olumeto olumn (olume- Column dimension- "artile sie- "artile sie distribution- "aAing density- "ore sie of the partiles- Flow rate- $isosity of the sampleand bu<er
$esolution@ degree o&
![Page 48: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/48.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 48/78
$esolution@ degree o&separation
s L 9.> M two omponent are present ine@ual proportions
s an be inreased by N
- 2sing longer olumn
- Hel medium with smaller partiles andEor greaterseleti(ity
- !mall sample (olume
- Oow ow rate
![Page 49: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/49.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 49/78
Separation time
ange from a few minutes to manyhours
5ording to the di?ulty of the
separation, the gel medium, theolumn length, and the ow rate
+mpro(e the resolution also lead to
longer separation time
S l l
![Page 50: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/50.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 50/78
Sample #olume
esolution depend onthe ratio of sample(olume to olumn
(olume
Oarge sample toolumn (olume ratiogi(ing lower resolution
$eomended sample #olume as a
![Page 51: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/51.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 51/78
$eomended sample #olume as aper ent o& the total bed #olume
&or good resolution
Medium $eomended sample#olume 3A t5
!ephade
!epharose!epharose CO!epharyl !uperde prepgrade
!uperose prepgrade!uperde!uperose
= P >
= P >= P >9 P =9 P =9 P =
&.>&.>
Moleular 'eight
![Page 52: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/52.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 52/78
Moleular 'eightdetermination
5 gel should be hosen, so that thesampleJs epeted ;# falls on the linearpart of the seleti(ity ur(e and in the
middle of a suitable range of alibrationstandard
+f the ;# is unAnown, a gel with widefrationation range '!epharyl ) will be
most suitable
2el fltration
![Page 53: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/53.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 53/78
media seletionguide
![Page 54: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/54.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 54/78
2 l Filt ti M di
![Page 55: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/55.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 55/78
2el Filtration Media
![Page 56: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/56.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 56/78
+on9e<hange 30dsorption5 hromatography
!eparation depends upon the re(ersibleadsorption of harged solute moleules toimmobilied ion ehange groups ofopposite harge.
"robably the most fre@uently used for theseparation and puriation of proteins,polypeptides, nulei aids,polynuleotides, and other harged
biomoleules.#idespread appliability, its high resol(ingpower, its high apaity, and the simpliityand ontrollability of the method.
+on9e<hange
![Page 57: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/57.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 57/78
ghromatogra
phy
![Page 58: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/58.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 58/78
+on /hangeD +nteration based on o(erall
harge 'less spei thana?nity)
D Cation ehange
D 5nion ehange
![Page 59: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/59.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 59/78
![Page 60: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/60.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 60/78
+on-/hange
![Page 61: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/61.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 61/78
+on /hangehromatography
+f p mobile phase :7.=
4hen harge of the proteinsN '-) '-) 'Q)
'Q)
-- Q Q
Q
Q
Q
Q
Q
Q-
--
--
-Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
5nion ehange olumn : Q harged
F ti l d i
![Page 62: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/62.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 62/78
Funtional groups used on ionehangers
Step'ise elution &or ion e<hange
![Page 63: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/63.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 63/78
Step'ise elution &or ion e<hange
0Cnity hromatography
![Page 64: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/64.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 64/78
0Cnity hromatography
!eparates proteins on the basis of a re(ersible
interation between a protein 'or group ofproteins) and a spei ligand oupled to ahromatography matri.
Can be used to separate ati(e biomoleules from
denatured or funtionally di<erent forms, toisolate pure substanes present at lowonentration in large (olumes of rude sampleand also to remo(e spei ontaminants
igh seleti(ity, hene high resolution, andusually high apaity for the protein's) of interest.
"uriation an be in the order of se(eralthousand-fold and reo(eries of ati(e material
are generally (ery high.
0Cnity
![Page 65: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/65.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 65/78
hromatography
5? it Ch t h
![Page 66: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/66.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 66/78
5?nity ChromatographyD2ses spei binding properties of moleulesEproteins
D!tationary phase has a polymer that an be o(alently linAedto a ompound alled a ligand that speially binds toprotein
0Cnity hromatography
![Page 67: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/67.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 67/78
0Cnity hromatographyseparation by biologial
binding interations
wash
porous
bead
glutathione
elute
GST apply sample
thrombin siteprotein of interest
/ampleN 2ST 92lutathione
H!4-tagged proteins bindto gluthatione on beads
on-speially orweaAly
bound proteins washedo<
H!4-tagged proteinseluted
with glutathione
Protein purifation by hromatography
![Page 68: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/68.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 68/78
Protein purifation by hromatography
What sort o& e%uipment do
![Page 69: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/69.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 69/78
% p'e need
![Page 70: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/70.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 70/78
-o' to pak the olumn
![Page 71: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/71.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 71/78
-o' to pak the olumn
5 puriation table for a
![Page 72: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/72.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 72/78
5 puriation table for ahypothetial enyme
Separating and #isuali)ing proteins
![Page 73: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/73.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 73/78
Separating and #isuali)ing proteinsSDS9P027/ Sodium dodeyl sul&ate polyarylamide gel eletrophoresis
9. eat sample with !6! and β -meraptoethanol
!6! : 6etergent 'anioni)- 6enatures proteins- Coats proteins- /ah protein has similar massEharge ratio
β-meraptoethanolE644- redues disulde bonds
=. !eparate on polyarylamide gel
- polymer of arylamineEbis-arylamide- 4/;/6, ammonium persulfate atalyst for polymeriation- "rotein migrates through gel matri in eletri eld.
7stimating the moleular
![Page 74: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/74.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 74/78
g'eight o& protein
Protein Properties
![Page 75: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/75.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 75/78
Protein Properties
• Proteins denatured by heating them in a sample buffer
containing SDS• The proteins no longer have any secondary, tertiary, or
quaternary structure
• esultant proteins taAe on a rod-liAe shape and auniform negati(e harge- to-mass ratio proportional to
their moleular weights
![Page 76: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/76.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 76/78
4he e@uipment of !6!
![Page 77: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/77.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 77/78
4he e@uipment of !6!-"5H/
"reparing gel
![Page 78: 1. Separasi Dan Purifikasi](https://reader034.vdocuments.us/reader034/viewer/2022050720/5695d0621a28ab9b0292427f/html5/thumbnails/78.jpg)
7/23/2019 1. Separasi Dan Purifikasi
http://slidepdf.com/reader/full/1-separasi-dan-purifikasi 78/78
"reparing gel