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Immunological MethodsImmunological MethodsImmunological MethodsImmunological Methods
Summer RainesSummer RainesSeptember 30th, 2009September 30th, 2009Bi h i t 660Bi h i t 660 smraines@wisc.edusmraines@wisc.edu
AttieAttie LabLabBiochemistry 660Biochemistry 660
OutlineOverview of Antibodies
St tStructureClassesModificationsModifications
Immunological TechniquesWestern BlottingImmunoprecipitationImmunofluorescence
Immunocytochemistry, ImmunohistochemistryEnzyme Linked ImmunoSorbant AssayEnzyme-Linked ImmunoSorbant AssayRadioimmunoassay
Antibody Structurey2 light chains (κ, λ) 2 heavy chains (α δ ε γ μ)2 heavy chains (α, δ, ε, γ, μ)
B d d b di lfidFab
Bonded by disulfides, non-covalent interactions
Variable regions recognize antigen
Fcantigen
Constant regions determineConstant regions determine isotype/function
Technical TerminologyTechnical Terminology
AntigenAntigen“Insulin”
1o Antibody“Mouse anti-insulin IgG”
2o Antibody“Goat anti-mouse IgG”
Polyclonal vs. Monoclonal A tib diAntibodies
antigen
1
34
21
23
Isolate Spleen cells +
Hybridize and select hybridoma clones
Isolate
4
B-cells Myeloma Cells
12
34
1
YAb-1
Ab 1
Serum31
antigen
1
2
34 YY
Ab-3
Ab-4
Ab-1
Ab-2
Ab-3
Ab-1 Ab-2 Ab-3 Ab-4
2
YAb-2
Ab 4
Ab-4Polyclonal Monoclonal
Polyclonal vs. Monoclonal ib d d CAntibody Pros and Cons
Pol clonals MonoclonalsPolyclonalsResistant to epitope
confirmation
MonoclonalsHighly reproducibleSpecificconfirmation
InexpensiveRelatively rapid
SpecificLimitless supply
Technically easy Sensitive to epitope conformation
Low reproducibilityCross-ReactiveLimited supply
ExpensiveTime-consumingTechnically difficultLimited supply Technically difficult
Antibody ModificationsAntibody Modifications
YYBiotin
YStrep
YFluorophores BiotinFluorophores
fluorescein, rhodamine, phycoerythrin
Y YBeads/Solid support
Y YEnzymesBeads/Solid support
agarose, magnets, ProteinAEnzymes
AP, HRP
Western BlottingWestern Blotting1D method to identify a specific protein from a
complex mixturecomplex mixture
Bl k
Transfer to membrane
Visualize
Block
BSA, milk, casein,
Tween-20membrane
1o Antibody
2o Antibodyy
SDS-PAGE to separate
Denatured antigen
Western BlottingWestern Blotting
Anti-ProteinX
Anti-ProteinY
Adapted from Christofk et al. (2008) Nature 452: 230-234
ImmunoprecipitationImmunoprecipitationIsolate antigen in solution for further analysis
SDS-PAGE
Western Blot
Mass Spec
Antigen containing Add 1o antibodyAdd 1o antibodyAdd 1o antibodyAntigen-containing sample
Add 1o antibodyAdd 1o antibody
Add beads (ProteinA)Add 1o antibody
Add beads (ProteinA)
Pellet and remove supernatantNative antigen
(before SDS-PAGE)
Co-immunoprecipitationCo immunoprecipitationIsolate antigen complex in solution for further
analysisanalysis
SDS-PAGE
Western Blot
Mass Spec
Antigen containing Add 1o antibodyAdd 1o antibodyAdd 1o antibodyAdd 1o antibodyAntigen-containing sample Native antigen
(before SDS-PAGE)
Add 1o antibodyAdd 1o antibodyAdd 1o antibody
Add beads (ProteinA)
Add 1o antibody
Add beads (ProteinA)
Pellet and remove supernatant
Co-immunoprecipitationCo immunoprecipitation
Mock WT Mut1 Mut2
ProteinY Over-expression
Blot: anti-ProteinXImmunoprecipitation:
Anti-ProteinXBlot: anti-ProteinY
t ote
Blot: anti-ProteinYLysate
Adapted from Bennasser and Jeang. (2006) Retrovirology 3:95
Immunocytochemistryy yImage antigen in live or fixed cells
FixFormalin, PFA, glutaraldehyde,
methanol, acetone YY
Y1o Antibody2o Antibody
Cells plated on slides
ProtienX ProteinY Merged
“Native” Antigen
Immunohistochemistry
Fix
yImage antigen in fixed tissue
Formalin, PFA, glutaraldehyde,
methanol, acetone
1o Antibody2o Antibody
Tissue sections on slides
“Native” Antigen
Immunohistochemistryy
Nuclei (DAPI)
Non-diabetic Diabetic
( )β-cells (ProteinX)α-cells (ProteinY)
Enzyme-Linked ImmunoSorbant Assay (ELISA)(ELISA)
Quantitative detection of antigen or antibody from a complex mixturefrom a complex mixture
Sandwich Method
Y
S
Y
Coat wellY Y
Add antigenY
Add colored
Y
YAdd enzyme-linked
Y
Coat well with 1o
antibody
Add antigen (purified or mixture) substrate
y1o Ab
Native Antigen
ELISAELISAStandards
ProteinX-Sample 1
0.1 0.5 1.0 2.0 5.0 10.0
ProteinX-Sample 2
4
2.53
3.54
banc
e
0.51
1.52
2
1
Abs
orb
00 5 10 15
1
Concentration (ng/mL)
Variations on ELISAIndirect Method Competitive MethodCompetitive MethodCompetitive Method
OR Y
Y
Y
D ib d Detect antigen
YY YY Y
Detect antibody Detect antigen
React antigen1 with labeled antibody before adding to
React antigen1 with labeled antibody before adding to
React antigen1 with labeled antibody before adding to
ELISPOT Methody g
antigen2-labeled welly g
antigen2-labeled wellless color = better binding
to antigen1
y gantigen2-labeled well
Y Detect cells expressing or secreting antigen
An ELISA Example –Are You Pregnant?
http://www.whfreeman.com/kuby/con_index.htm?06
Radioimmunoassay (RIA)Radioimmunoassay (RIA)Quantitative detection of antigen from a
complex mixturecomplex mixtureLess radioactivity =
more unlabeled antigen present inantigen present in
sample
Coat wellY Y Y Y
Read in gamma Add mixture to wellYY
Mix saturating
Native Antigen
Coat well with 1o
antibody
gcounterMix saturating
amounts of 125I-labeled antigen and unlabeled sample
Common Questions AskedCommon Questions AskedWestern IP IF ELISA RIA
Protein Expression?
Protein up- or down- regulation?ote up o do egu at o
Sub-cellular localization?
Protein binding partners?
Post-translational modification?
Protein/Metabolic profile?
Disease diagnostics?Antibodies against foreign proteins?
Summary of Techniques
E ChHigh
th h t Q tit ti
Excellent Very good Good FairPoor
Easy Cheap through-put Quantitative
Western
Immunoprecipitation
Immunofluorescence
ELISA
Radioimmunoassay
Antibody SourcesAntibody Sources
CommercialCommercialMolecular Probes (http://probes.invitrogen.com)
Jackson Labs (www.jacksonimmuno.com)
BD Pharmigen (www.bdbiosciences.com)
Abcam (www.abcam.com)
ServicesServicesHybridoma/monoclonal productionCustom-made polyclonalsp y
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