title: Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Shingo
authors.initials: S
authors.lastname: Nishikori
authors.forename: Takamitsu
authors.initials: T
authors.lastname: Hattori
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Fuchs
authors.forename: Norihisa
authors.initials: N
authors.lastname: Yasui
authors.forename: John
authors.initials: J
authors.lastname: Wojcik
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.forename: Brian D
authors.initials: BD
authors.lastname: Strahl
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
mesh_terms: D000906:Antibodies / Q000145:classification / Q000276:immunology*; D000915:Antibody Affinity; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D006657:Histones / Q000276:immunology*; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D006657:Histones