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Easy detection of chromatin binding proteins by the histone association assay

Abstract

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and nonchromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.

Abbreviations

ChIP:

Chromatin Immunoprecipitation

HAA:

Histone Association Assay

McM10:

Minichromosome Maintenance Protein 10

Orc2:

Origin Recognition Complex protein 2

WCE:

Whole Cell Extract

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Correspondence to Anja-Katrin Bielinsky Ph.D..

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Ricke, R.M., Bielinsky, AK. Easy detection of chromatin binding proteins by the histone association assay. Biol. Proced. Online 7, 60–69 (2005). https://doi.org/10.1251/bpo106

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  • DOI: https://doi.org/10.1251/bpo106

Indexing terms

  • Chromatin
  • Cross-linking Reagents
  • Formaldehyde
  • Histones
  • Immunoprecipitation