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

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 10/2012; 424(5). DOI: 10.1016/j.jmb.2012.09.022
Source: PubMed


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.

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    • "nanomolar to low micromolar range (for example, two papers reported K D values of 0.2, 4, 60, 83, 520, 820, 1000, 2200, and 2700 nM for several representative commercial antibodies [Nishikori et al. 2012; Hattori et al. 2013]). As indicated by these numbers, the ranges of affinities of antibodies and HMIDs overlap, but in general , antibodies bind more strongly. "
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    ABSTRACT: Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies.
    Genome Research 10/2014; DOI:10.1101/gr.170985.113 · 14.63 Impact Factor
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    • "Previous studies have demonstrated that reader domains interact with preferred histone substrates at low micromolar affinity [27-32], a range equal to or weaker than antibody recognition, which varies from micromolar to nanomolar [11]. To test whether such micromolar affinity is adequate for enriching specific nucleosomal PTMs, we next assessed the ability of ATRX-ADD to discriminate PTMs on a folded nucleosome structure, consisting of reconstituted mononucleosomes bearing either H3Kc4me3 or H3Kc9me3 (chemical analogs of H3K4me3 and H3K9me3 that permit stoichiometric modification). "
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    ABSTRACT: Background Histone post-translational modifications (PTMs) are key epigenetic regulators in chromatin-based processes. Increasing evidence suggests that vast combinations of PTMs exist within chromatin histones. These complex patterns, rather than individual PTMs, are thought to define functional chromatin states. However, the ability to interrogate combinatorial histone PTM patterns at the nucleosome level has been limited by the lack of direct molecular tools. Results Here we demonstrate an efficient, quantitative, antibody-free, chromatin immunoprecipitation-less (ChIP-less) method for interrogating diverse epigenetic states. At the heart of the workflow are recombinant chromatin reader domains, which target distinct chromatin states with combinatorial PTM patterns. Utilizing a newly designed combinatorial histone peptide microarray, we showed that three reader domains (ATRX-ADD, ING2-PHD and AIRE-PHD) displayed greater specificity towards combinatorial PTM patterns than corresponding commercial histone antibodies. Such specific recognitions were employed to develop a chromatin reader-based affinity enrichment platform (matrix-assisted reader chromatin capture, or MARCC). We successfully applied the reader-based platform to capture unique chromatin states, which were quantitatively profiled by mass spectrometry to reveal interconnections between nucleosomal histone PTMs. Specifically, a highly enriched signature that harbored H3K4me0, H3K9me2/3, H3K79me0 and H4K20me2/3 within the same nucleosome was identified from chromatin enriched by ATRX-ADD. This newly reported PTM combination was enriched in heterochromatin, as revealed by the associated DNA. Conclusions Our results suggest the broad utility of recombinant reader domains as an enrichment tool specific to combinatorial PTM patterns, which are difficult to probe directly by antibody-based approaches. The reader affinity platform is compatible with several downstream analyses to investigate the physical coexistence of nucleosomal PTM states associated with specific genomic loci. Collectively, the reader-based workflow will greatly facilitate our understanding of how distinct chromatin states and reader domains function in gene regulatory mechanisms.
    Epigenetics & Chromatin 04/2014; 7(1):7. DOI:10.1186/1756-8935-7-7 · 5.33 Impact Factor
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    ABSTRACT: Antibodies that recognise posttranslational modifications of histones play a central role in epigenetic proteomic research. Modification-specific antibodies are employed in chromatin immunoprecipitation, for Western blotting and during the immunoprecipitation steps for mass spectrometry-based global proteomic analysis. Knowledge about the antibodies' off-target binding is essential for the interpretation of experimental data. To address this challenge we developed a fast and cost efficient system for generating peptide bead arrays. We employed this method to establish a bead-based peptide array containing 384 peptides displaying phosphorylated, acetylated, methylated and citrullinated N-terminal regions of histone H2A, H2B, H3 and H4 and controls. We profiled the binding of 40 post-translational modification-specific antibodies important for epigenetic proteomic research.
    Proteomics 03/2013; 13(6). DOI:10.1002/pmic.201200383 · 3.81 Impact Factor
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