Sedimentation and immunoprecipitation assays for analyzing complexes that repress transcription

Ping Lu, Bruce S. Hostager, Paul B. Rothman, John D. Colgan

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationGene Regulation
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages365-383
Number of pages19
ISBN (Print)9781627032834
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume977
ISSN (Print)1064-3745

Keywords

  • Co-immunoprecipitation
  • Co-repressor proteins
  • DNA-binding proteins
  • Histone deacetylases
  • Immunoblots
  • Sucrose gradient sedimentation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Lu, P., Hostager, B. S., Rothman, P. B., & Colgan, J. D. (2013). Sedimentation and immunoprecipitation assays for analyzing complexes that repress transcription. In Gene Regulation: Methods and Protocols (pp. 365-383). (Methods in Molecular Biology; Vol. 977). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-284-1_30