title: Sedimentation and immunoprecipitation assays for analyzing complexes that repress transcription.
abstract: Co-repressor proteins function as platforms for the assembly of multi-subunit complexes that mediate transcriptional repression. Common components of such complexes are histone deacetylases, which catalyze the removal of acetyl groups from the tails of histones within nucleosomes, resulting in chromatin compaction and gene repression. In addition, co-repressor complexes generally interact with sequence-specific DNA-binding proteins that direct association with regulatory elements in the genome. Thus, identifying proteins that stably associate with co-repressors can provide insights regarding the biochemical function and target gene specificity of these molecules. Here, we describe a density gradient fractionation method for determining whether a co-repressor is incorporated into high-molecular complexes within cells and for identifying potential constituents of these complexes. We also describe a co-immunoprecipitation assay for confirming and further studying interactions between co-repressors and other proteins that may represent functional partners.
authors: Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors: Ping
authors: P
authors: Lu
authors: Bruce S
authors: BS
authors: Hostager
authors: Paul B
authors: PB
authors: Rothman
authors: John D
authors: JD
authors: Colgan
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002457:Cell Extracts / Q000302:isolation & purification; D002458:Cell Fractionation; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods*; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D009687:Nuclear Proteins / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000302:isolation & purification*; D014158:Transcription, Genetic
chemical_list: D002457:Cell Extracts; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D012097:Repressor Proteins