---
title: "How to Validate Histone PTM Antibodies for CUT&RUN"
slug: "how-to-validate-histone-ptm-antibodies"
description: "Learn how to validate histone PTM antibodies for CUT&RUN by assessing specificity, controls, cross-reactivity, and sequencing results."
tags: ["cut and tag", "cut and run", "cut and run-em"]
updated: 2026-02-27T15:26:39Z
published: 2026-02-27T15:26:39Z
canonical: "support.epicypher.com/how-to-validate-histone-ptm-antibodies"
---

> ## 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.

# How to validate histone PTM antibodies

> This article also applies to Multiomic CUT&RUN.

Validate histone PTM antibodies for CUTANA™ assays following the guidelines below, and read this blog post for [additional information](https://www.epicypher.com/resources/bloghow-to-select-the-best-histone-ptm-antibody-for-cutrun-and-cuttag/). 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](https://www.epicypher.com/antibodies-complete-list/) 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](https://www.epicypher.com/contact-us/) for recommendations or take the following steps to validate your own antibody.

1. **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
2. **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](https://www.epicypher.com/products/nucleosomes/snap-cutana-k-metstat-panel) to control reactions to gauge experimental success (see [this article](/v1/docs/using-snap-cutana-spike-in-controls-to-determine-assay-success)).
  - 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](/v1/docs/using-snap-cutana-spike-in-controls-for-histone-ptm-antibody-validation) for guidance.
  - **NOTE**: for labile histone PTMs, such as histone lysine acetylation, lightly cross-linking cells may help stabilize marks and improve assay performance.
3. **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](/v1/docs/analyzing-snap-cutana-spike-in-controls-and-expected-results).
4. **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.