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Sedimentation and Immunoprecipitation Assays for Analyzing Complexes that Repress Transcription

  • Ping Lu
  • Bruce S. Hostager
  • Paul B. Rothman
  • John D. Colgan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 977)

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.

Key words

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

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Ping Lu
    • 1
  • Bruce S. Hostager
    • 1
  • Paul B. Rothman
    • 1
  • John D. Colgan
    • 1
  1. 1.Department of Internal Medicine, Roy J. and Lucille A. Carver College of MedicineUniversity of IowaIowa CityUSA

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