Protocol

Affinity Purification of Interacting Proteins from Cell Lysates

This protocol was adapted from "Characterization of Protein Complexes," Chapter 10, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

In this protocol, recombinant protein or a chemically synthesized bioactive fragment is immobilized on resin and used as a probe to capture interacting proteins directly from a cell extract. Affinity-purified proteins are fractionated by gel electrophoresis and visualized by Coomassie staining. Proteins that interact specifically are identified by comparing this gel profile to one obtained from cell lysates passed over a control resin lacking the immobilized probe protein. Specific bands are excised from the gel, proteolytically digested in situ, and analyzed by liquid chromatography/tandem mass spectrometry to derive primary sequence information and consequently identify the interacting protein.