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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.

SUBMITTER: Nishikori S

PROVIDER: S-EPMC3502729 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Nishikori Shingo S Hattori Takamitsu T Fuchs Stephen M SM Yasui Norihisa N Wojcik John J Koide Akiko A Strahl Brian D BD Koide Shohei S

Journal of molecular biology 20121002 5

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 PTM ...[more]

PMID: 23041298

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Project description:Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly.

Dataset Information

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

Publications

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

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