General approach for characterizing in vitro selected peptides with protein binding affinity

Andrew C. Larsen, Annabelle Gillig, Pankti Shah, Sujay P. Sau, Kathryn E. Fenton, John C. Chaput

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


In vitro selection technologies are important tools for identifying high affinity peptides to proteins of broad medical and biological interest. However, the technological advances that have made it possible to generate long lists of candidate peptides have far outpaced our ability to characterize the binding properties of individual peptides. Here, we describe a low cost strategy to rapidly synthesize, purify, screen, and characterize peptides for high binding affinity. Peptides are assayed in a 96-well dot blot apparatus using membranes that enable partitioning of bound and unbound peptide-protein complexes. We have validated the binding affinity constants produced by this method using known peptide ligands and applied this process to discover five new peptides with nanomolar affinity to human α-thrombin. Given the need for new analytical tools that can accelerate peptide discovery and characterization, we feel that this approach would be useful to a wide range of technologies that utilize high affinity peptides.

Original languageEnglish (US)
Pages (from-to)7219-7223
Number of pages5
JournalAnalytical Chemistry
Issue number15
StatePublished - Aug 5 2014

ASJC Scopus subject areas

  • Analytical Chemistry


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