Validate histone PTM antibodies for CUTANA™ assays following the guidelines below, and read this blog post for additional information. Using highly specific antibodies will make a huge difference in the biological accuracy and quality of your results. EpiCypher is committed to offering the best histone PTM antibodies for your CUTANA studies, so you can be confident in your data. If we do not offer an antibody to your target of interest, contact us for recommendations or take the following steps to validate your own antibody.
Obtain 3-5 antibodies (preferably monoclonal) to your PTM from various vendors. Make sure that the antibodies target distinct epitopes.
EpiCypher scientists have observed that antibodies good for immunofluorescence (IF) applications tend to produce good data in our CUTANA assays. Although using IF-validated antibodies is NOT a guarantee for success, it may help guide antibody selection for targets that lack validated reagents
Perform CUTANA assays with all candidate antibodies. Additional controls and recommendations:
Always include reactions with H3K4me3 and H3K27me3 positive control antibodies and IgG negative control antibodies reactions. Add the SNAP-CUTANA™ K-MetStat Panel to control reactions to gauge experimental success (see this article).
For antibodies to lysine methylation PTMs (H3K4, H3K9, H3K27, H3K36, or H4K20 me1, me2, and me3), add the SNAP-CUTANA K-MetStat Panel to reactions to confirm on-target specificity. See this article for guidance.
NOTE: for labile histone PTMs, such as histone lysine acetylation, lightly cross-linking cells may help stabilize marks and improve assay performance.
Confirm positive and negative controls show expected sequencing results, including data from the SNAP-CUTANA K-MetStat Panel.
The negative controls should have low, nonspecific recovery of nucleosomes from the K-MetStat Panel, while the positive control reaction should only recover spike-in nucleosomes carrying the target PTM (e.g. H3K4me3 with less than 20% cross-reactivity to off-targets).
Positive controls should also generate robust peaks in expected genomic regions (i.e. sharp peaks at active transcription start sites for H3K4me3).
See an expanded discussion and example data here.
Examine sequencing data and compare the profiles from target antibodies as follows:
Lysine methylation PTMs: Antibodies should show <20% cross-reactivity using the SNAP-CUTANA K-MetStat Panel. Confirm that the genomic enrichment is consistent with the target PTM biology (e.g. broad vs narrow peak at functional elements) and shows high signal-to-noise.
For other PTMs: Compare results and select a specific antibody based on yields, expected target enrichment, and signal-to-noise in sequencing data.