An assessment of histone-modification antibody quality

Abstract

We have tested the specificity and utility of more than 200 antibodies raised against 57 different histone modifications in Drosophila melanogaster, Caenorhabditis elegans and human cells. Although most antibodies performed well, more than 25% failed specificity tests by dot blot or western blot. Among specific antibodies, more than 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use, and we provide a website for posting new test results (http://compbio.med.harvard.edu/antibodies/).

Figure 1

Representative western assays and results. (a) Western blot of anti-H3K4me2 (Millipore, 07-030, lot DAM1543701), anti-H3S10ph (Wako, 303-35199), and anti-H4K20me3 (Diagenode, CS-057, lot A9-002). Left panel shows Coomassie blue stained gel of worm nuclear extract and recombinant H3 (Active Motif, 31207), showing the amount of protein loaded in each lane and approximately equal levels of histone H3 in the nuclear extract and recombinant H3 sets of lanes. Histone H3 is marked with an arrowhead and histone H4 with an asterisk. Anti-H3K4me2 passed, because it recognized only H3 in the nuclear extract and not unmodified H3. Anti-H3S10ph failed, because it recognized unmodified H3 with equal intensity to H3 in the nuclear extract. Anti-H4K20me3 failed, because it recognized non-histone proteins and perhaps H3 instead of H4 in nuclear extract. All western blot images are available at http://compbio.med.harvard.edu/antibodies/. Images are also available at http://www.modencode.org/docs/hmav.html (worm and fly) http://epigenome.ucsd.edu/antibodies.html (human). (b) Summary of fly and worm western blot results. Antibodies against core histones were not included in the summation, since they are expected to detect recombinant histones. Test results differed among groups for 3 antibodies (pass versus no signal, or fail versus no signal). Those antibodies were included in the pass or fail categories, respectively. (c) Performance of antibodies tested in fly and worm nuclear extracts (nuc. ext.). Antibody results were binned into 5 mutually exclusive groups; the percentage is plotted, with the number of antibodies shown above each bar. Same exceptions were applied as in panel b. (c) Performance of antibodies tested in human whole-cell extracts (WCE). Many antibodies classified as “Histone + other bands” passed ChIP tests.

Figure 2

Representative dot blot assays and results. (a) Dot blot characterization of anti-H3K4me2 (Abcam, ab32356, lot 577702) and anti-H3K27ac (Abcam, ab4729, lot 726657). Top panel shows the positions of histone tail peptides spotted on membranes. Anti-H3K4me2 passed. Anti-H3K27ac failed due to detection of multiple peptides. Human, fly, and worm dot blot images are available at the web sites noted in the legend to Figure 1. (b) Summation of peptide blot results. 149 antibodies were classified as described in the text. Low signal indicates that only the highest peptide concentration was detected by the antibody. See Supplementary Fig. 1 for a description of the peptide array used for each antibody, and Supplementary Table 1 for enumeration of cross-reacting peptides.

Figure 3

ChIP-chip and ChIP-seq. (a) Representative ChIP-chip characterization. Shown is anti-H3K36me1 from two different sources (Abcam, ab9048 lot #18882 and H. Kimura, 1H1). A ~60 kb region of C. elegans chromosome IV is shown, with annotated genes (X-axis) and ChIP-chip z-scores (standardized log₂ ratios of ChIP/Input signals) (Y-axis) plotted for biological replicates using both antibodies. The replicates were highly correlated using the Abcam antibody (passed), but not using the 1H1 antibody (failed). (b) Summary of results. Antibodies against core histones were not included in the summation.