Immunoprecipitation techniques for the analysis of transcription factor complexes

Abstract

Interactions among transcription factors can be detected and analyzed by a variety of in vitro and in vivo approaches. In many studies, the existence of putative interactions among transcription factor partners is initially established from yeast two-hybrid screening and in vitro protein association analysis. The ability to detect candidate interacting proteins in coimmunoprecipitates from cell lysates provides an important criterion for establishing the authenticity of such protein interactions in vivo. This article describes methodology developed for detecting interactions between the helix–loop–helix protein, Id3, and the paired homeodomain protein, Pax5, and interactions involving the zinc finger transcription factor, CTCF. The importance of empirically establishing optimum conditions for cell lysis, selection of appropriate antibodies, conditions for immunoprecipitation, and detection of interacting partners are discussed.