EPITOPIC’s approach, searching for peptides binding to antibodies or other targets, is unique in saving time and materials by reducing wet lab procedures to a minimum and applying a statistical approach to analyse large data sets of peptide sequences obtained with NGS.
The procedure starts with capturing binders from a proprietary random library of 5×109 16-mer peptides. This naïve library can be used in all projects. It has the unique feature of a well-controlled distribution of individual amino acids. Even small deviations from the statistically expected frequency can be detected using a specially developed software package. Indeed a few hundred antibody molecules in the selection experiment are enough to identify pools of binding sequences in hundred thousand of sequences obtained by NGS. Please see Epitope Fingerprinting Process for details.
The library covers almost all possible 6-mer sequences including discontinuous variants, so aligning these candidate binders allows predicting for example not only an antibody’s epitope. It unravels also tolerated amino acid variations and conserved adjacent residues helping to understand potential binding in discontinuous epitopes. In contrast to approaches using simple linear peptides or libraries of peptides derived from the antigen, the library structure allows the selection of structurally constrained peptides without applying special libraries or time-consuming synthesis of peptides.
The variety of epitopes identified is well reflected in the following examples of different antibodies against the PTEN protein:
Catalog# 655002, Clone 4C11A11(BioLegend)
Biotinylated PTEN (D4.3) XP Rabbit mAb (CellSignalling)
PTEN antibody (PerkinElmer)