How do I validate a chromatin-associated protein antibody?

EpiCypher offers CUT&RUN antibodies to diverse chromatin-associated proteins, including transcription factors (e.g. CTCF), chromatin reader proteins (e.g. BRD4), chromatin modifying enzymes (e.g. MLL1), remodelers (e.g. SMARCA2 and SMARCA4), and commonly used epitope tags (e.g. HA).

Can't find the antibody you need? Follow the steps below or contact us for recommendations.

We recommend selecting the antibody that best balances the need for robust DNA yields, enrichment for expected sequence motifs and/or peak structures, and high signal over background. Testing native and lightly cross-linked cell samples is also ideal for protein targets.

1. Obtain 3-5 antibodies (preferably monoclonal) to your protein 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 CUT&RUN assays. Although using IF-validated antibodies is NOT a guarantee for success, it may help guide CUT&RUN antibody selection for targets that lack validated reagents.
2. Perform CUT&RUN with all candidate antibodies. Additional controls and recommendations:
   • Include reactions with positive and negative control antibodies (e.g. EpiCypher H3K4me3 SNAP-Certified Antibody and EpiCypher Rabbit IgG Antibody, respectively). Add the SNAP-CUTANA K-MetStat Panel to control reactions to gauge experimental success. See this article for guidance.
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.
4. Examine sequencing data. Select antibodies that generate high signal-to-noise in CUT&RUN and generate genomic distribution profiles consistent with the reported function of the target protein (for example, DNA binding motif analysis for transcription factors).