H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN

SKU: 13-0041
Pack Size: 100 µg
  • Type:  Mixed Monoclonal*
  • Host:  Rabbit
  • Applications:  CUT&RUN, ICC/IF, WB

    NOTE: Previous lots were validated in ChIP, however lots are no longer tested in this application.

  • Reactivity:  Human, Mouse, Drosophila, Yeast, Wide Range (Predicted)
  • Format:  Protein A affinity-purified
  • Target Size:  15 kDa


This H3K4me3 (histone H3 lysine 4 trimethyl) antibody meets EpiCypher’s lot-specific SNAP-Certified™ criteria for specificity and efficient target enrichment in CUT&RUN. This requires <20% cross-reactivity to related histone PTMs determined using the SNAP-CUTANA™ K-MetStat Panel of spike-in controls (EpiCypher 19-1002, Figure 1). High target efficiency is confirmed by consistent genomic enrichment at 500k and 50k starting cells (Figures 2-4). This antibody targets histone H3 trimethylated at lysine 4, which is enriched at active promoters near transcription start sites (TSS).

*Mixed Monoclonal: a pool of multiple recombinant monoclonal antibodies.

Validation Data

Figure 1: SNAP specificity analysis in CUT&RUN
CUT&RUN was performed as described in Figure 6. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the K-MetStat panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K4me3 set to 100%).

Figure 2: CUT&RUN genome wide enrichment
CUT&RUN was performed as described in Figure 6. Sequence reads were aligned to 18,793 annotated transcription start sites (TSSs ± 2 kbp). Signal enrichment was sorted from highest to lowest (top to bottom) relative to the H3K4me3 - 500k cells sample (all gene rows aligned). High, medium, and low intensity are shown in red, yellow, and blue, respectively. H3K4me3 antibody produced the expected TSS enrichment pattern, which was consistent between 500k and 50k cells and greater than the IgG negative control.

Figure 3: H3K4me3 CUT&RUN representative browser tracks
CUT&RUN was performed as described in Figure 6. Gene browser shots were generated using the Integrative Genomics Viewer (IGV, Broad Institute). Similar results in peak structure and location were observed for both cell inputs.

Figure 4: Immunofluorescence
Representative images (60X magnification) of HeLa cells fixed, permeabilized, and immunostained to show endogenous localization of H3K4me3. (A) H3K4me3 antibody (green, 1:100 dilution) detected with an Alexa Fluor® 488 conjugated anti-rabbit secondary. (B) DAPI stained nuclei (blue). (C) Rhodamine stained cytoskeletal F-actin (red). (D) A composite of panels a, b, & c demonstrating nuclear localization of H3K4me3. (E) Negative control lacking H3K4me3 primary antibody to assess background.

Figure 5: Western blot data.
Representative Western data of H3K4me3 in whole cell extracts from HeLa, Hep G2, HCT 116, MCF7, U-2 OS, A549, HEK-293, NIH/3T3, and PC-12 cells. 30 µg of lysate was resolved via SDS-PAGE and detected with H3K4me3 antibody at a 1:250 dilution.

Figure 6: CUT&RUN methods
CUT&RUN was performed on 500k and 50k K562 cells with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) spiked-in prior to the addition of 0.5 μg of either H3K4me3 or IgG negative control (EpiCypher 13-0042) antibodies. The experiment was performed using the CUTANA™ ChIC/CUT&RUN Kit v3 (EpiCypher 14-1048). Library preparation was performed with 5 ng of CUT&RUN enriched DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Both kit protocols were adapted for high throughput Tecan liquid handling. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Reaction sequencing depth was 8.3 million reads (IgG 500k cell input), 15.5 million reads (IgG 50k cell input), 9.8 million reads (H3K4me3 500k cell input) and 8.6 million reads (H3K4me3 50k cell input). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.

Technical Information

A synthetic peptide corresponding to histone H3 trimethylated at lysine 4
Stable for 1 year at -20°C from date of receipt
Protein A affinity-purified antibody in PBS pH 7.4, 0.09% sodium azide
Target Size
15 kDa

Recommended Dilution

0.5 µg per reaction
Immunofluorescence (IF):
Western Blot (WB):

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