How to assess Histone antibody specificity and quality

The specificity of antibodies to histone modifications (or any antigen for that matter) is crucial for obtaining meaningful experimental data. Cross-reactivity to similar modifications at other residues (e.g. H3K27Me3 vs H3K9Me3) or off-target recognition of other modification isoforms (e.g. H3K27Me1 vs H3K27Me3) can seriously compromise the integrity of any study using these types of reagents.

Epicypher's Histone Peptide Array technology

Fig. 1: Histone Peptide Array schematic representation. Source: EpiCypher.

Rothbart and colleagues have used a unique quantitative histone peptide array to investigate the specificity of more than 100 commercially available histone modification antibodies, raised against 43 unique modifications at 33 distinct residues (Fig. 1).

Quantitative ChIP-based array (ICe-ChIP) and Histone Antibody Specificity Database

Chromatin ImmunoPrecipitation (ChIP) is a key technique for the epigenetic and chromatin biology. ChIP requires the use of high quality antibodies which have have been tested on this novel quantitative ChIP-based array called ICe-ChIP.

Their findings were surprising (and more than a little disconcerting), in that many of the antibodies tested exhibited a large degree of off-target recognition.

For example, over 40% of the “Methyl-specific” antibodies recognized a lower or higher state of methylation in addition to the nominal modification.

The study also highlighted the long-suspected influence (either positive or negative) of neighbouring modifications on antibody recognition (e.g. can H3S10P influence binding to H3K9Me).

All these data are available as a community resource via the Histone Antibody Specificity Database interactive web-based tool. (Fig. 2)

 EpiCypher’s EpiTitan Histone Array

The Histone Antibody Specificity Database is an interactive resource to curate histone antibody specificity data.

Fig. 2: Histone Antibody Specificity Database – Interactive resource to curate histone antibody specificity data, such as generated with the EpiTitan Histone Peptide Array. Left Panel – the main icon allowing selection of which histone you want to view antibody data for. Right Panel – bar graph showing specificity data for the Histone H3K4Me3 Antibody Cat. nr 23613-0004.

Rothbart’s work is based on EpiCypher’s EpiTitan Histone Array. It highlights the need to carefully determine the specificity of your histone modification antibodies prior to performing any experiments.

To know more about this publication:

  • Rothbart et al. “An Interactive Database for the Assessment of Histone Antibody Specificity” (2015) Molecular Cell 59, 502–511 .

Thanks to EpiCypher for sharing this application of their arrays with us.

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.