--- title: "Validate histone PTM antibodies using SNAP-CUTANA™ Spike-ins" slug: "using-snap-cutana-spike-in-controls-for-histone-ptm-antibody-validation" description: "Learn how SNAP-CUTANA™ Spike-ins validate histone PTM antibody specificity and identify off-target binding in CUT&RUN and CUT&Tag." updated: 2024-10-01T17:16:12Z published: 2024-10-01T17:16:12Z canonical: "support.epicypher.com/using-snap-cutana-spike-in-controls-for-histone-ptm-antibody-validation" --- > ## Documentation Index > Fetch the complete documentation index at: https://support.epicypher.com/llms.txt > Use this file to discover all available pages before exploring further. # Validate histone PTM antibodies using SNAP-CUTANA™ Spike-ins At EpiCypher, we have developed two main applications of the SNAP-CUTANA™ Spike-in Controls for CUTANA CUT&RUN and CUT&Tag assays. The first is determination of workflow success, discussed [here](/v1/docs/using-snap-cutana-spike-in-controls-to-determine-assay-success). The other application of SNAP-CUTANA Spike-ins is for histone PTM antibody validation. ## **Why test histone PTM antibodies?** 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](https://www.epicypher.com/products/nucleosomes/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](https://chromatinantibodies.com/). ![snap chip@2x](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/snap%20chip@2x.png) **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](https://pubmed.ncbi.nlm.nih.gov/30244833/). These results were a major driving factor in the development of [SNAP-CUTANA Spike-in technology](https://www.epicypher.com/technologies/snap-spike-in-controls). To ensure the success of emerging CUTANA™ epigenomic assays, defined controls are needed to find the best antibodies possible. ## **Why use SNAP-CUTANA Spike-ins vs. other antibody validation methods?** This strategy is the only method that directly confirms antibody specificity in CUTANA assays 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 CUTANA assays! ## **Metrics for SNAP-CUTANA antibody validation in CUTANA assays** EpiCypher is using these defined nucleosome spike-in controls to identify best-in-class histone PTM antibodies for CUTANA assays. We offer a collection of [SNAP-Certified Antibodies for CUT&RUN](https://www.epicypher.com/products/antibodies/cutana-cut-and-run-antibodies/histone-ptms) 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 ## **How 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](https://www.epicypher.com/products/nucleosomes/snap-cutana-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 CUTANA assays using the recommended amount of cells or nuclei in each reaction. - Add the SNAP-CUTANA Spike-in Panel to CUTANA reactions just before addition of primary antibody, as indicated in the protocol. Include control reactions with the K-MetStat Panel to confirm workflow success. See [this article](/v1/docs/using-snap-cutana-spike-in-controls-to-determine-assay-success) for an overview. - Aim for <20% antibody cross-reactivity and consistent genomic enrichment with 500,000 cells (or 100,000 nuclei for CUT&Tag). - In CUT&RUN, to help ensure high antibody efficiency, validation at 50,000 cells is also recommended. ## Additional notes - 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](https://www.epicypher.com/products/nucleosomes/snap-cutana-k-metstat-panel).