Histone

Histone H3K4me3 Antibody, SNAP-Certified™ for CUT&RUN and ChIP

$475.00
SKU: 13-0041
Pack Size: 100 µg
  • Type:  Mixed Monoclonal*
  • Host:  Rabbit
  • Applications:  CUT&RUN, ChIP, ICC/IF, WB
  • Reactivity:  Human, Mouse, Drosophila, Yeast, Wide Range (Predicted)
  • Format:  Protein A affinity-purified
  • Target Size:  15 kDa

Description

This antibody meets EpiCypher’s lot-specific SNAP-Certified™ criteria for specificity and efficient target enrichment in both CUT&RUN and ChIP applications. 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). In ChIP, this requires <20% cross-reactivity to related histone PTMs and >5% of target input recovered as determined using the SNAP-ChIP® K-MetStat Panel of spike-in controls (EpiCypher 19-1001, Figure 7) [1].

*Mixed Monoclonal: a pool of multiple monoclonal antibodies.

Validation Data

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 v3.0 (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). Sample sequencing depth was 6.7 million reads (IgG 50k cell input), 11.5 million reads (IgG 500k cell input), 10.2 million reads (H3K4me3 50k cell input) and 16.7 million reads (H3K4me3 500k cell input). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions

Figure 1: SNAP specificity analysis in CUT&RUN
CUT&RUN was performed as described above. 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 above. 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 - 50k 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 above. 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: Antibody efficiency analysis in CUT&RUN using cell input correlation
CUT&RUN was performed as described above. Genome-wide correlation analysis was performed to compare H3K4me3 antibody enrichment using 500k and 50k cell inputs. The log of the number of reads per 75 bp binned region across the genome is plotted for both samples. CUT&RUN data generated using this H3K4me3 antibody are highly correlated between the two cell inputs (Pearson correlation r = 0.970), indicating high efficiency of H3K4me3 antibody target recovery.

Figure 5: 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 6: Western blot data
Western analysis 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 7: SNAP-ChIP-qPCR specificity and enrichment analysis
Histone H3K4me3 antibody (5 µg) was tested in a native ChIP experiment using chromatin from K562 cells (5 µg) with the SNAP-ChIP K-MetStat Panel (EpiCypher 19-1001) spiked-in prior to micrococcal nuclease digestion. Specificity (left y-axis) was determined by qPCR for the DNA barcodes corresponding to modified nucleosomes in the SNAP-ChIP panel (x-axis). Black bar represents antibody efficiency (right y-axis; log scale) and indicates percentage of the target immunoprecipitated relative to input. Error bars represent mean ± SEM in replicate ChIP experiments.

Technical Information

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

Recommended Dilution

CUT&RUN:
0.5 µg per reaction
ChIP:
2 - 5 µg per 5 µg chromatin
Immunofluorescence (IF):
1:100
Western Blot (WB):
1:250

References

Background References:
[1] Shah et al. Mol. Cell (2018). PMID: 30244833

Product References:

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