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Mol Cell Proteomics DOI:10.1074/mcp.M111.015941

Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

Publication TypeJournal Article
Year of Publication2012
AuthorsPeach, SE, Rudomin, EL, Udeshi, ND, Carr, SA, Jaffe, JD
JournalMol Cell Proteomics
Date Published2012 May
KeywordsAcetylation, Antibodies, Antibody Specificity, Chromatin Immunoprecipitation, Cluster Analysis, HeLa Cells, Histones, Humans, Methylation, Phosphorylation, Protein Binding, Protein Processing, Post-Translational

The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.


Alternate JournalMol. Cell Proteomics
PubMed ID22442256
PubMed Central IDPMC3418838
Grant ListR21 DA025720 / DA / NIDA NIH HHS / United States