Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the "reproducibility crisis," demonstrating the need for rigorous, platform-appropriate validation.
Pubmed ID: 30244833 RIS Download
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Software tools for Motif Discovery and next-gen sequencing analysis. Used for analyzing ChIP-Seq, GRO-Seq, RNA-Seq, DNase-Seq, Hi-C and numerous other types of functional genomics sequencing data sets. Collection of command line programs for unix style operating systems written in Perl and C++.
View all literature mentionsA generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms.
View all literature mentionsSystem that classifies genes by their functions, using published scientific experimental evidence and evolutionary relationships to predict function even in absence of direct experimental evidence. Orthologs view is curated orthology relationships between genes for human, mouse, rat, fish, worm, and fly.
View all literature mentionsSystem that classifies genes by their functions, using published scientific experimental evidence and evolutionary relationships to predict function even in absence of direct experimental evidence. Orthologs view is curated orthology relationships between genes for human, mouse, rat, fish, worm, and fly.
View all literature mentionsThis polyclonal targets Histone H3 (tri methyl K4)
View all literature mentionsThis polyclonal targets H3K4me1
View all literature mentionsThis polyclonal targets H3K4me3
View all literature mentionsThis unknown targets Tri-Methyl-Histone-H3 (Lys4)
View all literature mentionsThis unknown targets Trimethyl-Histone H3 (Lys4)
View all literature mentionsThis polyclonal targets H3K4me3
View all literature mentionsThis monoclonal targets Human Histone H3, trimethyl (Lys4)
View all literature mentionsThis monoclonal targets H3K4me3
View all literature mentionsThis polyclonal targets Tri-Methyl-Histone H3 (Lys4)
View all literature mentionsThis unknown targets H3K4me3
View all literature mentionsThis monoclonal targets H3K4me3
View all literature mentionsThis unknown targets Di-Methyl-Histone H3 (Lys4)
View all literature mentionsThis recombinant polyclonal targets H3K4me2
View all literature mentionsThis unknown targets H3K4me2
View all literature mentionsThis monoclonal targets H3K4me2
View all literature mentionsThis polyclonal targets H3K4me2
View all literature mentionsThis monoclonal targets H3K4me2
View all literature mentionsThis unknown targets H3K4me1
View all literature mentionsThis recombinant polyclonal targets H3K4me1
View all literature mentionsThis unknown targets Monomethyl-Histone H3 (Lys4)
View all literature mentionsThis polyclonal targets H3K4me1
View all literature mentionsThis polyclonal targets H3K4me1
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View all literature mentionsA technical computing software and computing environment that provides users with algorithms and mathematical functions for various projects and purposes. The resource incorporates other Wolfram products such as Wolfram Algorithmbase, Wolfram Language, and Wolfram Knowledgebase.
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View all literature mentionsCell line K-562 is a Cancer cell line with a species of origin Homo sapiens (Human)
View all literature mentionsThis polyclonal targets H3K4me3
View all literature mentionsThis polyclonal targets Histone H3 (tri methyl K4)
View all literature mentionsThis polyclonal targets H3K4me1
View all literature mentions