Broad Ranges of Affinity and Specificity of Anti-Histone Antibodies Revealed by a Quantitative Peptide Immunoprecipitation Assay

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 bottlen...

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Bibliographic Details
Published in:Journal of molecular biology 2012-12, Vol.424 (5), p.391-399
Main Authors: Nishikori, Shingo, Hattori, Takamitsu, Fuchs, Stephen M., Yasui, Norihisa, Wojcik, John, Koide, Akiko, Strahl, Brian D., Koide, Shohei
Format: Article
Language:English
Subjects:
Antibodies - classification
Antibodies - immunology
Antibody Affinity
antibody–antigen interaction
binding capacity
chromatin
Chromatin Immunoprecipitation - methods
dissociation
epigenetics
flow cytometry
histone code
histones
Histones - immunology
monoclonal antibodies
peptides
precipitin tests
quantitative analysis
Sensitivity and Specificity
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Summary: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. [Display omitted] ► Limited characterization of antibodies is a bottleneck in epigenetics research. ► We describe a method that quantitatively determines affinity, specificity and binding capacity. ► The method mimics the ChIP format and highly sensitive. ► Commercial “ChIP-grade” antibodies show large ranges in their performance.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2012.09.022