Protocol

Phage-Display Methodology for the Study of Protein-Protein Interactions: Stage 3, Binding Analysis

This protocol was adapted from "Phage-Display Methodology for the Study of Protein-Protein Interactions," Chapter 9, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

This method for creation of phage libraries can be used for the generation and/or engineering of variant protein domains. It was adapted in our laboratory for the development of variant domains of SpA (the virulence factor, protein A, produced by clinical isolates of Staphylococcus aureus) with novel Fab-binding specificities. In Stage 3 of the method, described here, the rounds of selection are analyzed to evaluate the enrichment for binders. To do this, polyethylene glycol (PEG)-precipitated phage from each round of panning are tested in an assay, be it ELISA-based or flow cytometric. The assay should include all rounds of phage samples, from the initial unselected phage to the last round and helper phage (as a negative control). The method chosen to evaluate whole phage libraries can also be used as a system of evaluating single clones from the appropriate round. The appropriate round is the one that yields a library exhibiting the best binding characteristics (or best signal in the detection assay) associated with the protein-protein interaction. This protocol outlines a basic ELISA-based analysis strategy.