SNAP-ChIP Antibody Validation*

Histones are decorated by post-translational modifications (PTMs) that serve as epigenetic signatures for gene expression. Chromatin immunoprecipitation (ChIP) is a common application to examine these PTMs at individual loci across the genome. Peptide array has traditionally been used for determining the capability of these antibodies and assesses the antibodies’ ability to specifically recognize only the peptides containing the targeted modification. However, peptide array does not recapitulate antibody performance in a ChIP experiment because of the requirement for the antibody to recognize the modified histone in a nucleosome context. EpiCypher Inc. has the SNAP-ChIP™ (Sample Normalization and Antibody Profiling) method for normalizing ChIP experiments that can be used to test antibody specificity. Spiking in a panel of recombinant semi-synthetic modified nucleosomes allows one to determine the efficiency of the immunoprecipitation and if an antibody is enriching the target of interest compared to other histone PTMs (i.e., off-target) in the panel.

EpiCypher is partnering exclusively with Thermo Fisher Scientific to create best-in-class ChIP antibodies for histone PTMs using SNAP-ChIP spike-in control panels to validate specificity. The goal of this partnership is to create a portfolio of SNAP-ChIP-validated histone PTM antibodies; providing researchers a new level of confidence in the data they generate by creating a new standard for specificity in ChIP antibody validation.

SNAP-ChIP validated antibodies

In the example below, SNAP-ChIP K-MetStat™ (EpiCypher, 19-1001) was performed to analyze the histone specificity of H3K36me3 Recombinant Rabbit Monoclonal Antibody (RM155) (Cat. No. MA5-24687) via ChIP. The ChIP assay spikes in a panel of post-translationally modified, DNA-barcoded, semi-synthetic nucleosomes during the normal ChIP workflow.

Figure 1. SNAP-ChIP K-MetStat for H3K36me3. The panel includes un-, mono-, di-, and tri-methyl forms of H3K4, H3K9, H3K27, H3K36, and H4K20 nucleosomes that can later be quantified to determine how much of each PTM is immunoprecipitated in the ChIP reaction. H3K36me3 Recombinant Rabbit Monoclonal Antibody (RM155) (Cat. No. MA5-24687) was tested in native ChIP with 3 µg chromatin from HEK-293 cells. Specificity (left y-axis; all bars mean 177 SEM from six independent ChIP experiments) was determined by quantitative real-time PCR (qPCR) to each modified nucleosome in the SNAP-ChIP K-MetStat panel (x-axis). The black bar represents antibody efficiency (right y-axis; log scale) and indicates percentage of the barcoded H3K36me3 nucleosome target immunoprecipitated relative to input.

In another example, SNAP-ChIP K-AcylStat™ (EpiCypher, 19-1001) was performed to analyze the histone specificity of H3K27ac Monoclonal Antibody (Cat. No. MA5-23516) in ChIP. The ChIP assay spikes in a panel of post-translationally modified, barcoded, semi-synthetic nucleosomes during the normal ChIP workflow.

Figure 2. SNAP-ChIP K-AcylStat for H3K27ac. The panel includes unmodified control plus nucleosomes with single acylations on H3, as well as combinatorial PTMs that can later be quantified to determine how much of each PTM is immunoprecipitated in the ChIP reaction. H3K27ac Monoclonal Antibody (Cat. No. MA5-23516) was tested in native ChIP with 3 µg K-652 cell chromatin. Specificity (left y-axis) was determined by quantitative real-time PCR (qPCR) to each modified nucleosome in the SNAP-ChIP K-AcylStat panel (x-axis). Black bar represents antibody efficiency (right y-axis; log scale) and indicates percentage of the barcoded H3K27ac nucleosome target immunoprecipitated relative to input.

Invitrogen antibodies that have been verified using SNAP-ChIP are indicated with a “verified specificity” symbol in search results and on relevant product pages. The data showing the verification will be provided on each product page.