PEPperPRINT released two new application notes on the use of the PEPperCHIP® Peptide Microarrays. The first application note describes the permutation scan of two known epitopes, HA and M13, for the in-depth analysis of epitopes with respect to sequence conservation, the actual motif length and the possible contribution of variable positions to antibody binding (see also Epitope Substitution Scan). The permutation scans were based on peptide microarray in which each amino acid position of the starting epitope is gradually exchanged by the 19 other main amino acids as well as two additional D-amino acids to further identify new epitope variants as well as to investigate the incorporation of non-natural amino acids for e.g. higher in-vivo stabilities for peptide-based immunizations and vaccinations. Staining with monoclonal anti-HA and polyclonal anti-M13 antibodies revealed well-defined staining patterns that enabled an in-depth view on epitope-antibody interactions.
For the first time, the second application note covers an enzymatic assay based on the multiplexed mapping of various tyrosine kinase substrates on PEPperCHIP® Peptide Microarrays. In general, the mapping of kinase substrates provides useful information for monitoring kinase specific activities in cells or tissue samples, or for setting up and optimizing kinase activity assays. Ten sets based on known and generic tyrosine substrates were permutated around the Y-phosphorylation sites resulting in a peptide microarray with 1,209 different substrate variants printed as duplicates. Two copies of the peptide microarray were assayed with two cytosolic tyrosine kinases c-Src and Csk to enable an in-depth characterization of phosphorylation sites and kinase substrate specificities. We observed a thorough consistency of the microarray experiments with published data, a pronounced similarity of both c-Src and Csk, but also some clear differences in substrate specificities.
Both application notes are available as download including all peptide microarray data in the Application Note section.