Tuesday, June 14, 2011

Antibody Epitope Mapping Protocol

Antibody Epitope Mapping Protocol Review
Visit www.arrayit.com for more Information.
A library of peptides corresponding to the test is first synthesized. The peptides can be up to 20 amino acids long with any length overlap desired covering the entire test antigen sequence. The peptides are dissolved in 100% DMSO to a final concentration of 10 mg/ml and printed at a final concentration of 5 mg/ml in 50% DMSO onto SuperEpoxy 2 Microarray Substrates using a NanoPrint Microarrayer.  Let printed substrates dry over-night and then block and wash according to the standard processing protocol for SuperEpoxy 2 Substrates.  Store processed microarrays clean and dry at room temperature,  Use vacuum desiccation if they are to be stored for long periods of time.
In order to determination of binding specificity of antibodies to the peptide library, dilute each antibody to be tested at in Protein Microarray Reaction Buffer with sufficient volume to react all the spots on the microarray for 60 min at RT a concentration of up to 3 ยต g/ml can be used, much lower concentrations also work such as 50 ng/ml.  Arrayit sells various tools and equipment to make this process easy.  Please see:
After primary antibody reaction, wash 3 times each for 3 minutes in Protein Microarray Wash Buffer and 1 time each PBS for 1 minute and dry with a microarray centrifuge.  Then proceed to react microarrays with appropriate secondary antibodies conjugated to dyes compatible with a microarray scanner.  Alternatively react with biotinylated secondary antibodies and stain with a 1 to 2000 dilution of 1 mg/ml streptavidin conjugated to a fluorescent dye compatible with a slide based microarray scanner.  React 60 min at RT in Protein Microarray Reaction Buffer and repeat wash steps and final rinse in high purity PBS. Scan on microarray scanner, such as Arrayit Innoscan 710 and quantify the median fluorescence intensity measurement of antibody binding to individual peptide sequences on the microarray.
Contact Todd Martinsky todd@arrayit.com for more information