Investigations of transcriptional regulation and chromatin organization often rely heavily on antibodies to various post-translational modifications on histones. The reproducibility and biological relevance of histone-modification studies depends on the specificity and performance of the antibodies, most of which are now provided commercially. As part of our activities in the NIH modENCODE and Roadmap Reference Epigenome initiative, we are using antibodies in conjunction with chromatin immunoprecipitation (ChIP) to determine the genomic distributions of histone modifications in C. elegans, Drosophila, and human cells. We have tested the specificity and utility of 246 antibodies raised against 3 unmodified histones and 57 different histone modifications (Egelhofer et al. Nature Struc & Molec Biol 18: 91-93). Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot analysis. Among specific antibodies, more than 20% failed in ChIP experiments. We discuss our tests of antibody specificity and our criteria for successful performance. We advise rigorous testing of histone-modification antibodies in the organism of interest before use, and we provide a website for posting new results
(http://compbio.med.harvard.edu/antibodies/).