November 20, 2008

Validation of Commercial Histone Modification Antibodies to be Usedfor ChIP
Gary Karpen, Susan Strome
Overview: There are numerous issues that need to be addressed with respect to validating
commercial histone modification antibodies used extensively for ChIP-chip and ChIP-seq
analyses. The main issues are 1) specificity of antibodies with respect to other nuclear/chromatinproteins, 2) specificity with respect to unmodified histones and other modified histone residues(e.g. H3K9me and H3K27me), 3) specificity with respect to mono-, di-, and tri- methylation atthe same residue (e.g. H3K9me1, me2 and me3), and 4) lot-to-lot variation. Validation ofcommercial antibodies is necessary to produce the high quality data sets desired by themodENCODE and ENCODE consortia. We propose that all commercial histone antibodies bevalidated by at least 2 independent methods, as described below. New lots of antibody must beanalyzed independently. The proposed tests may be performed by ENCODE/modENCODE labs
or by the companies that sell the antibodies, but only if the companies provide data for thespecific lots of antibody.
Test #1: All antibodies must be checked for reactivity with non-histone proteins and withunmodified histone by performing Western blot analysis of total nuclear extract and recombinanthistone. To enable visual quantification of reactivity, a concentration series of both extract andrecombinant histone should be analyzed, using recombinant histone levels that are comparable tothe respective histone in nuclear extract. Since cross-reactivity may vary between species, thistest should be performed using nuclear extracts from each species to be studied by ChIP. Innuclear extracts, the specific nuclear histone band should constitute at least 50% of the nuclear
protein signal, show at least 10-fold enrichment relative to any other single band, and show atleast 10-fold enriched signal relative to unmodified histone.
Test #2 options: In addition to test #1, antibody specificity must be verified by at least one
additional test. The tests and the pros and cons of each are described below, followed by thelikely flow of tests.
A. Peptide binding tests. Histone tail peptides with particular modifications can bepurchased commercially. Peptide binding and peptide competition assays provide a fast
method to initially evaluate the specificity and relative strength of binding of antibodies tohistone tails with different modifications (e.g. H3K9 and H3K27 and me1, me2, and me3
levels of methylation). A potential drawback is that antibodies may differ in their binding
specificity toward histone tail peptides in vitro versus toward full-length histones in thecontext of chromatin in IP experiments. Nevertheless, observing at least a 10-fold enrichedbinding signal for the modification of interest relative to other modifications would
contribute to confidence in antibody specificity.
B. Mass spec. For antibodies generated against related and historically problematicmodifications, the ability of the antibody to effectively distinguish between similar histone
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marks (e.g. H3K9me and H3K27me) and between different levels of methylation (e.g.
H3K9me1, me2 and me3) should be tested by mass spec analysis of material IP'd from
histone preparations. The target modification should constitute at least 80% of the IP'dhistone signal, and contaminating bands should not contribute more than 20%.
C. Mutants defective in modifying histones. Mutants (in S. cerevisiae, S. pombe,
Drosophila, C. elegans, or mammalian cells) that lack the ability to catalyze particular
histone modifications offer the opportunity to test antibody specificity. Antibody signal
should be reduced to below 10% of wild-type signal in mutant samples, compared to wildtype. RNAi depletion of histone modifying activity may be substituted for mutants. Mutantor RNAi reduction of signal can be assayed by Western blot analysis or byimmunofluorescence staining. Mutant/RNAi tests usually don't allow testing antibodies forthe ability to discriminate between mono, di, and trimethylation. In cases where more than
one enzyme modifies the same residue (e.g. K9 methylation in Drosophila), double mutants
or RNAi may be required.
D. Mutant histones. Mutant histones (e.g. histone H3 with Lys4 mutated to Arg or Ala)
expressed in yeast provide another avenue to alter histones for testing specificity by western
blot analysis or even by ChIP. Since the modified residue is mutated, we expect at least a 10fold reduction in signal relative to wild-type histone preparations. Mutant histone tests
cannot distinguish whether antibodies discriminate between mono, di, and trimethylation.
Flow of tests: For antibodies to methylated histone residues, we envision the flow of tests willbe: #1 western blot analysis to make sure the antibody does not show significant cross-reaction
with unmodified histone or non-histone proteins; #2A peptide binding/competition tests to makesure the antibody does not interact with histone tail peptides lacking modifications or bearing
other modifications; for similar and problematic modifications, #2B mass spec analysis to makesure the antibody does not IP unmodified histone or histone bearing other modifications.
For antibodies to histone modifications other than methylations, we envision the flow oftests will be: #1 western blot analysis to make sure the antibody does not show significant crossreaction with unmodified histone or non-histone proteins; #2A peptide binding/competition tests
to make sure the antibody does not interact with histone tail peptides lacking modifications orbearing other modifications OR one of the other #2 tests listed above.
Grandfathering old antibodies and testing new lots of antibodies: Once ChIP-chip or ChIPseq results are obtained with antibodies validated as described above, then old or new antibodies
can be validated by similar testing or by obtaining ChIP results using old/new antibodies that arestatistically consistent with ChIP results using validated antibodies (based on ENCODE andmodENCODE standards for statistical agreement between replicates).
Use of 2 different antibodies: Even if antibodies pass the specificity tests described above,observing very similar ChIP results with 2 independent antibodies would give added confidence
in the results. Therefore, we encourage using 2 independent antibodies whenever possible,
providing statistical comparisons of the results, and presenting the intersection of the peak setsobtained with the 2 antibodies.

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