Histone H3K4me3 Antibody: SNAP-ChIP® Certified, CUTANA™ CUT&RUN Compatible

SKU: 13-0041
Pack Size: 100 µg
Type:  Recombinant Polyclonal* Host:  Rabbit
Mol Wgt.:  15 kDa Appl.:  ChIP, ChIP-Seq,CUT&RUN, L
Format:  Aff. Pur. IgG Reactivity: H, M, WR
Histone H3K4me3 Antibody: SNAP-ChIP Certified, CUTANA™ Compatible Description: This antibody meets EpiCypher’s “SNAP-ChIP® Certified” criteria for specificity and target enrichment in ChIP (<20% cross-reactivity to related histone post-translation modifications and >5% recovery of target input determined using SNAP-ChIP K-MetStat Panel spike-in controls; EpiCypher Catalog No. 19-1001). Although its specificity in CUT&RUN has yet to be empirically determined in situ using spike-in controls, CUT&RUN data produced by this antibody shows a genome-wide enrichment pattern characteristic of H3K4me3 and is highly correlated with ChIP-seq (Figures 3-5).
*Recombinant polyclonal: pool of multiple monoclonals

Histone H3K4me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible Certified Immunogen: A synthetic peptide corresponding to histone H3 trimethylated at lysine 4.

Histone H3K4me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible Certified Formulation: Protein A affinity-purified antibody in PBS, pH 7.2 with 0.09% sodium azide.

Histone H3K4me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible Certified Storage and Stability: Stable for 1 years at -20°C from date of receipt.

Histone H3K4me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible Certified Application Note:
Recommended Diltutions:

2 - 5 μg per 5 µg chromatin
0.5 µg (1.0 µL) per sample
1:250 - 1:4000 dilution

Histone H3K4me3 Antibody, SNAP-ChIP Certified, CUTANA™ Compatible References:

Grzybowski et al (2015) Mol Cell 58:886

Shah et al (2018) Mol Cell 72:162

View technical datasheet for this product. 13-0041 Datasheet

Applications Key: ChIP-Chromatin IP; E-ELISA; FACS-Flow cytometry; IF-Immunofluorescence; IHC-Immunohistochemistry; ICC-Immunocytochemistry; L-Luminex; IP-Immunoprecipitation; WB-Western Blotting

Reactivity Key: B-Bovine; Ce-C. elegans; Ch-Chicken; Dm- Drosophila; Eu-Eukaryote; H-Human; M-Mouse; Ma-Mammal; R-Rat; Sc-S.cerevesiae; Sp-S. pombe; WR-Wide Range (predicted); X-Xenopus; Z-Zebrafish

13-0041 Luminex Data

Luminex Mutliplexed Specificity Profiling: Histone H3K4me3 antibody was assessed using a Luminex® based approach employing dCypher® Nucleosome K-MetStat Panel (EpiCypher Catalog No. 16-9002). The panel comprises biotinylated designer nucleosomes (x-axis) individually coupled to color coded Luminex Magplex® beads. Antibody binding to the panel of 16 nucleosomes was tested in multiplex at a 1:1000 dilution, and detected with second layer anti-IgG*PE. Data was generated using a Luminex FlexMAP3D®. Data normalized to relevant on-target (H3K4me3; set to 100)
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13-0041 SNAP-ChIP qPCR Data

SNAP-ChIP Data: Histone H3K4me3 antibody (5 µg) was tested in a native ChIP experiment using chromatin from K-562 cells (5 µg) with the SNAP-ChIP K-MetStat Panel (EpiCypher Catalog No. 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.
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13-0041 Representative Sequencing Tracks

H3K4me3 SNAP-ChIP-seq and CUT&RUN representative tracks: Gene browser shots generated using the Integrative Genomics Viewer (IGV, Broad Institute) show representative loci for H3K4me3 ChIP-seq (blue track, 5 μg antibody) and CUT&RUN (green track, 1:100 antibody dilution). For comparison H3K4me2 ChIP-seq is shown (top red track, EpiCypher Catalog No. 13-0027) as well as an ENCODE H3K4me3 ChIP-seq track using a different antibody (bottom orange track, GEO accession number GSM733680). (A) A broad genome window shows similar results for all three approaches. (B) Antibody cross-reactivity differences are illustrated at the putative CELSR2 enhancer region (gray boxed region), which is enriched in H3K4me2 (top red track) but not H3K4me3 (Shah et al., Mol Cell 2018). EpiCypher H3K4me3 antibody lacks a peak in this region in contrast to the ENCODE antibody known to cross-react with H3K4me2 (Shah et al., Mol Cell 2018). This result is consistent with the hypothesis that the EpiCypher antibody specifically enriches H3K4me3 in both ChIP-seq and CUT&RUN. Methods: Native ChIP-seq was performed as described (Shah et al., Mol Cell 2018). CUT&RUN was performed using EpiCypher CUTANA pAG-MNase for ChIC/CUT&RUN (EpiCypher Catalog No. 15-1016) as described (EpiCypher.com/cutana-protocol). Library preparation was performed with 10 ng DNA using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina®. ChIP libraries were sequenced on an Illumina NextSeq 550 (2x150bp paired end). The total number of reads was 46.5 million for H3K4me3 ChIP-seq and 4.3 million for CUT&RUN.
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13-0041 Genome Wide Analysis

ChIP-seq and CUT&RUN genome wide analysis EpiCypher H3K4me3 antibody (Catalog No 13-0041) was tested in native ChIP-seq (A) and CUT&RUN (B) using the methods described above. Genome-wide analysis of H3K4me3 enrichment (signal intensity) flanking annotated transcription start sites (TSSs; +/- 3kb) is graphed as a cumulative histogram plot (top) and shown in a heatmap (bottom). Individual gene loci in each row of the heatmap are colored by signal intensity and sorted by strongest to lowest enrichment (top to bottom). EpiCypher H3K4me3 antibody displays a characteristic enrichment pattern proximal to TSSs in both ChIP-seq and CUT&RUN.
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13-0041 Correlation Analysis

ChIP-seq vs. CUT&RUN correlation analysis Genome-wide correlation analysis was performed to compare EpiCypher H3K4me3 antibody (Catalog No 13-0041) enrichment in ChIP-seq and CUT&RUN. The number of reads per 500 bp binned region across the genome is plotted for ChIP-seq (x-axis) vs. CUT&RUN (y-axis) (EaSeq). ChIP-seq and CUT&RUN data generated using this antibody are highly correlated (Pearson correlation r = 0.811).
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