Histone H3K27me3 Antibody, SNAP-ChIP® Certified, CUTANA™ CUT&RUN Compatible - DISCONTINUED

SKU: 13-0030
Pack Size: 100 μg
Type:  Monoclonal Host:  Rabbit
Mol Wgt.:  15 kDa Appl.:  ChIP, ChIP-Seq, CUT&RUN, Luminex, IF, IHC, WB
Format:  Aff. Pur. IgG Reactivity: H, M, WR

This antibody has been discontinued. For alternative antibody options, please visit https://www.epicypher.com/snap-chip-certified-antibodies/.

Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible Description: This antibody meets EpiCypher’s “SNAP-ChIP® Certified” criteria for specificity and target enrichment in ChIP (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 H3K27me3 and is highly correlated with ChIP-seq (Figures 3-5).
Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible Immunogen: A synthetic peptide corresponding to histone H3 trimethylated at lysine 27.

Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible Formulation: Protein A affinity-purified antibody (1 mg/mL) in PBS, with 0.09% sodium azide, 1% BSA, and 50% glycerol.

Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible Storage and Stability: Stable for 1 years at -20°C from date of receipt.

Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible Application Notes Recommended dilutions:
ChIP/ChIP-seq: 2 - 5 μg per 1x106 cells CUT&RUN: 1:100 Luminex: 1:4000 IF/IHC: 0.5 - 2 μg/mL WB: 1 - 2 μg/mL
Histone H3K27me3 Antibody, SNAP-ChIP Certified, CUTANA CUT&RUN Compatible References:
Grzybowski et al (2015) Mol Cell 58:886
Shah et al (2018) Mol Cell 72:162

View technical datasheet for this product. 13-0030 Datasheet

Applications Key: ChIP-Chromatin IP; E-ELISA; FACS-Flow cytometry; IF-Immunofluorescence; IHC-Immunohistochemistry; 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

13-0030 Luminex Data

Luminex multiplexed specificity profiling: H3K27me3 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 1:4000 dilution, and detected with second layer anti-IgG*PE. Data was generated using a Luminex FlexMAP3D®. Data is normalized to target (H3K27me3; set to 100).
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13-0030 SNAP-ChIP data

SNAP-ChIP-qPCR specificity and enrichment analysis: H3K27me3 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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13-0030 Sequencing Tracks

H3K27me3 SNAP-ChIP-seq and CUT&RUN representative tracks: A gene browser shot generated using the Integrative Genomics Viewer (IGV, Broad Institute) shows a representative locus for EpiCypher H3K27me3 ChIP-seq replicate experiments (blue tracks, 5 μg antibody) and CUT&RUN (green track, 1:100 antibody dilution). For comparison ENCODE H3K27me3 ChIP-seq using a different antibody is shown (bottom orange track, GEO accession number GSM733658). Similar results in peak structure and location were observed throughout the genome for EpiCypher H3K27me3 antibody in 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 37.8 and 34.9 million for the ChIP-seq replicates and 8.0 million for CUT&RUN.
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13-0030 Genome Wide Analysis

ChIP-seq and CUT&RUN genome wide analysis: EpiCypher H3K27me3 antibody was tested in native ChIP-seq A and CUT&RUN B using the methods described above. Genome-wide analysis of H3K27me3 enrichment (signal intensity) flanking annotated transcription start sits (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 H3K27me3 an2body displays a characteristic enrichment pattern downstream of the TSS, remaining elevated throughout the body of the gene.
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13-0030 Correlation Analysis

ChIP-seq vs. CUT&RUN correlation analysis: Genome-wide correlation analysis was performed to compare EpiCypher H3K27me3 antibody enrichment in ChIP-seq and CUT&RUN. The number of reads per 10 kb binned region across the genome is plotted for CUT&RUN (x-axis) vs. ChIP-seq (y-axis) (EaSeq). ChIP-seq and CUT&RUN data generated using this antibody are highly correlated (Pearson correlation r = 0.864).
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13-0030 Western Blot

Western Blot Analysis: Recombinant histone H3.3 (Lane 1) and acid extracts of HeLa cells (Lane 2) were blotted onto PVDF and probed with 1 µg/mL EpiCypher H3K27me3 Antibody.
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13-0030 Immunohistochemistry

Immunohistochemistry (IHC): IHC staining of HepG2 cells using EpiCypher H3K27me3 Antibody.
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