Using SNAP-CUTANA Spike-in Controls for histone PTM antibody validation
At EpiCypher, we have developed two main applications of the SNAP-CUTANA Spike-in Controls for CUT&RUN. The first is determination of workflow success, discussed here. The other application of SNAP-CUTANA Spike-ins is for histone PTM antibody validation.
Why is testing histone PTM antibodies important?
Histone PTM antibodies are notoriously nonspecific, which can result in misleading biological interpretations. EpiCypher is well aware of this problem. We first developed SNAP nucleosome spike-in technology for ChIP-seq (SNAP-ChIP Spike-ins) and used them to test more than 400 lysine-methylation and lysine-acylation antibodies in ChIP assays. We found that >70% of histone PTM antibodies show significant off-target binding and/or low binding efficiency – even highly cited antibodies! The results of this study can be found at chromatinantibodies.com.
Figure 1. A highly cited H3K4me3 antibody fails SNAP Spike-in testing and shows extensive cross-reactivity to H3K4me2 (middle panel). Highly specific H3K4me2 (top panel; orange) and H3K4me3 (bottom panel; blue) antibodies are shown for comparison. See Shah et al. 2018.
These results were a major driving factor in the development of SNAP-CUTANA Spike-in technology. To ensure the success of emerging CUT&RUN (and CUT&Tag) epigenomic assays, defined controls are needed to find the best antibodies possible.
Why should I use SNAP-CUTANA Spike-ins to validate antibodies, compared to other methods?
This strategy is the only method that directly confirms antibody specificity in CUT&RUN against physiological on- and off-target substrates. EpiCypher knows from experience that histone PTM antibodies do not perform the same across assays – for instance, a good ChIP antibody is NOT guaranteed to work in CUT&RUN!
EpiCypher metrics for SNAP-CUTANA CUT&RUN antibody validation:
EpiCypher is using these defined nucleosome spike-in controls to identify best-in-class histone PTM antibodies for CUT&RUN. We offer a collection of SNAP-Certified Antibodies for CUT&RUN and can provide antibody recommendations upon request.
We validate antibody specificity as well as efficiency, allowing users to be confident when using reduced cell numbers. Each of our SNAP-Certified™ Antibodies show:
- High specificity: <20% recovery of off-target PTMs
- High target efficiency: Robust profiling at 500,000 and 50,000 starting cells
To use SNAP-CUTANA Spike-ins for PTM antibody testing:
- Purchase a SNAP-CUTANA Panel that contains your histone PTM target. For instance, you can use the K-MetStat Panel for any reaction that is mapping a histone methyl-lysine mark included in the panel.
- Source several antibodies to your histone PTM target to test, ideally from several sources and/or targeting distinct epitopes.
- Test antibodies side-by-side in CUT&RUN using 500,000 native cells per reaction.
- Add the SNAP-CUTANA Spike-in Panel to CUT&RUN reactions just before addition of primary antibody, as indicated in the protocol. Include control reactions with the K-MetStat Panel to confirm workflow success.
- Aim for <20% antibody cross-reactivity and consistent genomic enrichment with 500,000 cells. To help ensure high antibody efficiency, validation at 50,000 cells is also recommended.
- Do NOT add a SNAP-CUTANA Panel to reactions targeting a protein or a PTM that is not represented in the panel.
- Note that for CUTANA CUT&RUN Kit users, this will require purchase of additional K-MetStat Panel.