Immunoprecipitation techniques for the analysis of transcription factor complexes.
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.
Klenova
Elena
E
Department of Biological Sciences, Gene Regulation Laboratory, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK.
Chernukhin
Igor
I
Inoue
Toshiaki
T
Shamsuddin
Shaharum
S
Norton
John
J
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002457:Cell Extracts; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D046911:Macromolecular Substances; D009363:Neoplasm Proteins / Q000378:metabolism; D051757:PAX5 Transcription Factor; D011233:Precipitin Tests / Q000379:methods*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism*
D000906:Antibodies; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D051796:Inhibitor of Differentiation Proteins; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D014157:Transcription Factors; C075640:ID3 protein, human