Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay

Shingo Nishikori, Takamitsu Hattori, Stephen M. Fuchs, Norihisa Yasui, John Wojcik, Akiko Koide, Brian D. Strahl, Shohei Koide

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Antibodies directed against histone posttranslational modifications (PTMs) are critical tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. However, a lack of quantitative methods for characterizing such antibodies has been a major bottleneck in accurate and reproducible analysis of histone modifications. Here, we report a simple and sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Importantly, it determines the apparent dissociation constants for the interactions of an antibody with peptides harboring cognate or off-target PTMs. Analyses of commercial antibodies revealed large ranges of affinity, specificity and binding capacity as well as substantial lot-to-lot variations, suggesting the importance of quantitatively characterizing each antibody intended to be used in ChIP experiments and optimizing experimental conditions accordingly. Furthermore, using this method, we identified additional factors potentially affecting the interpretation of ChIP experiments.

Original languageEnglish
Pages (from-to)391-399
Number of pages9
JournalJournal of Molecular Biology
Volume424
Issue number5
DOIs
Publication statusPublished - Dec 14 2012
Externally publishedYes

Keywords

  • antibody-antigen interaction
  • epigenetics
  • flow cytometry
  • histone code

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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