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Life Science > Cell Biology > Antibodies > Antibodies Applications Center > Protocols > Epitope Mapping
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Epitope Mapping | ||
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Sigma® has prepared product lists for commonly used protocols from Cold Spring Harbor Protocols. |
View the protocol on BioSupplyNet.com.
Selected Protocol from "Using Antibodies," Ed Harlow and David Lane.
Epitope Mapping by Competition Assay
Ed Harlow and David Lane
ABSTRACT
The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can be used with antibodies that bind both conformational and linear epitopes, and it is most useful in the analysis of monoclonal antibody specificity because polyclonal sera typically recognize multiple different epitopes. This simple, ELISA-based protocol examines the ability of two hypothetical, newly derived monoclonal antibodies, XH1 and XH2, to compete with the well-characterized ZO-1 anti-YFP1 antibody for binding to the target antigen YFP1; hypothetical antibody PC10 is used as a control. First, 96-well plates are coated with the YFP1 antigen and direct binding assays are run to determine that all of the antibody preparations are active and able to react with the immobilized antigen. Second, the competition analysis itself is carried out. Note that the names of antibodies and antigens should be considered as generic.
| Protocol Material | Product No. | Description | Add to Cart |
| Buffers, Solutions, and Reagents | |||
| NaHCO3 | S5761 | Sodium bicarbonate, cell culture tested, insect cell culture tested |
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| PBS | P5368 | Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified |
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| PBS with BSA (1% [w/v]; should not contain azide) | P3688 | Phosphate buffered saline, pH 7.4, contains BSA, powder |
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| PBS with Tween 20 | P3563 | Phosphate buffered saline, pH 7.4, contains Tween 20, dry powder |
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| Rabbit anti-mouse IgG peroxidase conjugate | A9044 | Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in rabbit |
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| Streptavidin-peroxidase conjugate | S5512 | Streptavidin−Peroxidase from Streptomyces avidinii |
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| CaCl2 | C2661 | Calcium chloride, plant cell culture tested, anhydrous |
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| MgCl2 | M8266 | Magnesium chloride, anhydrous |
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| NaCl | S3014 | Sodium chloride, for molecular biology |
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| KCl | P9541 | Potassium chloride, for molecular biology |
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| Na2HPO4 | 71649 | Sodium phosphate dibasic dodecahydrate |
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| KH2PO4 | P9791 | Potassium phosphate monobasic, for molecular biology |
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| 3, 3', 5, 5'-tetramethylbenzidine (TMB) | T8665 | 3,3',5,5'-Tetramethylbenzidine (TMB) Liquid Substrate System |
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| Sodium acetate | S2889 | Sodium acetate |
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| Hydrogen peroxide (30%) | H1009 | Hydrogen peroxide solution, 30 % (w/w) in H2O |
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| H2SO4 | 380075 | Sulfuric acid, purified by double-distillation |
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| Water | W4502 | Water, Molecular Biology Reagent |
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.