Specificity Analysis of Antibodies That Recognize Histone Posttranslational Modifications

Hitting the mark with histone antibodies

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The nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It functions to package DNA into units of ~150 base pairs wrapped around two copies each of histones H2A, H2B, H3, and H4, while also significantly contributing to the regulation of gene expression. Histones have a remarkable assortment of posttranslational modifications (PTMs)—including methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, and ribosylation [1]. Due to the large number of modified histone residues, and the additional complexity resulting from the different methylation states of lysine or arginine residues, studying the epigenome requires a set of highly specific and validated tools [1].

Antibodies specific for histone PTMs are essential reagents in a variety of experimental techniques, including chromatin immunoprecipitation (ChIP), western blotting, immunofluorescence, and immunohistochemistry. ChIP is extensively used to assess protein–DNA interactions and to analyze the occupancy of chromatin modifications on a genome-wide scale. Because certain histone modifications may display similar DNA binding patterns, the accuracy of a ChIP experiment depends upon the specificity of the antibody and its ability to distinguish between subtly different PTMs, such as dimethylation versus trimethylation. Recent studies aimed at testing the quality of commercially available histone PTM antibodies have raised concerns regarding their specificity, which is of paramount importance when analyzing the association of histone modifications and disease [2-5]. Thus, rigorous specificity analysis and functional validation of histone PTM antibodies are needed.

Here we describe our analysis of Invitrogen histone PTM antibodies. We are conducting extensive specificity testing of our antibodies using peptide arrays and functional ChIP validation assays, and comparing their performance to that of other widely cited, commercially available histone PTM antibodies. Table 1 lists a set of recently tested Invitrogen histone PTM antibodies that performed as well as or better than corresponding histone PTM antibodies from other manufacturers in histone peptide arrays; a detailed explanation of how this comparison was performed is discussed in Figure 1.

Table 1. Invitrogen histone PTM antibodies validated on peptide arrays.

Methods for comparing histone PTM antibodies

To compare the specificity of antibodies for a particular histone PTM, we tested antibodies from various manufacturers using histone peptidearrays, which contain 384 peptides from the N-terminal tails of histones featuring 59 posttranslational modifications. This peptide array assay was followed by an analysis of the functional performance of the histone PTM antibody in ChIP. Commercially available antibodies were chosen based on citations and their applicability in peptide arrays, peptide dot blots, ChIP-qPCR, and ChIP-Seq, as well as in additional applications such as western blotting and immunocytochemistry.

Specificity analysis using peptide arrays

Figure 1A shows representative peptide arrays that have been incubated with either the Invitrogen Di-Methyl-Histone H3 (Lys4) Antibody (anti-H3K4me2 rabbit oligoclonal antibody) or a commercially available antibody from another supplier purported to have the same specificity. The Invitrogen anti-H3K4me2 antibody only binds to peptides that contain the H3K4me2 modification, whereas the other supplier’s antibody binds to peptides that contain the specified modification as well as peptides containing other modifications (Figure 1A). The data can also be represented as a graph of the “specificity factor” for each modification, which is the ratio of the average intensity of all spots containing a particular PTM to the average intensity of all spots lacking that PTM on the peptide array (Figure 1B). “Specific” antibodies are defined as those showing greater than a two-fold difference in the specificity factors for binding at the target site versus at the best nontarget site. Invitrogen antibodies that performed as well as or better than competitors showed equal or higher fold differences, respectively, between the specificity factors for binding at the target and at the best nontarget site (listed in Table 1).

Functional analysis in ChIP assays

Chromatin pull-down in a ChIP assay is evidence of the presence or absence of a specific histone PTM at a particular genomic locus. Use of a specific histone PTM antibody helps ensures that chromatin is not pulled down by nonspecific interactions. As this assay requires recognition of the modification in the context of nucleosomes, functional validation of a histone PTM antibody for use in ChIP is required to establish both that target epitopes are accessible and that the antibody is binding to expected loci.

Figure 1C shows the performance of the two anti-H3K4me2 antibodies in a ChIP assay. The Invitrogen anti-H3K4me2 antibody shows the expected enrichment of H3K4me2 on the active but not the silent loci, whereas the other supplier’s anti-H3K4me2 antibody shows much lower fold enrichment of the active loci as compared with the silent loci. These data clearly emphasize that antibodies for ChIP should be chosen not only for their ability to pull down chromatin but also for their ability to show enrichment of target histone PTMs at the expected genomic regions.

Find the right antibody for your experiments

When choosing a histone PTM antibody, two major considerations are the stringent verification of antibody specificity and the functional validation in ChIP. To address reports of the lack of reproducibility in commercially available histone PTM antibodies [2-5], histone PTM antibodies in the Invitrogen portfolio are undergoing extensive specificity verification and functional ChIP validation.* This comprehensive study underscores the breadth of histone PTM antibodies offered by Thermo Fisher Scientific and should serve as a useful guide when choosing the right antibody for a particular experiment.

* The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. The product(s) was not validated for clinical or diagnostic use.

References

1. Kouzarides T (2007) Cell 128:693–705. 
2. Fuchs SM, Strahl BD (2011) Epigenomics 3:247–249. 
3. Nishikori S, Hattori T, Fuchs SM et al. (2012) J Mol Biol 424:391–399. 
4. Egelhofer TA, Minoda A, Klugman S et al. (2011) Nat Struct Mol Biol 18:91–93. 
5. Peach SE, Rudomin EL, Udeshi ND et al. (2012) Mol Cell Proteomics 11:128–137.