IgG Affinity Capture of TAP-tagged Protein Complexes from Cell Extracts: Affinity Purification Step 1

Abstract

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the immunoglobulin G (IgG) affinity capture of TAP-tagged complexes. This procedure is followed by calmodulin affinity purification.