Histone

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

$475.00
SKU: 13-0041
Pack Size: 100 µg
  • Type:  Recombinant Polyclonal*
  • Target Size:  15 kDa
  • Format:  Aff. Pur. IgG
  • Host:  Rabbit
  • Appl.:  ChIP, ChIP-Seq, CUT&RUN, Luminex
  • Reactivity: H, M, WR

Description

This antibody meets EpiCypher’s “SNAP-ChIP® Certified” criteria for specificity and efficient 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, Figure 2). Although its specificity in CUT&RUN has yet to be 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

Validation Data

Figure 1: Luminex multiplexed 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 FlexMAP 3D®. Data is normalized to relevant on-target (H3K4me3; set to 100).

Figure 2: SNAP-ChIP-qPCR specificity and enrichment analysis
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.

Figure 3: 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). Both a broad genome view (A) as well as a close up at the GAPDH gene promotor (B) show that the EpiCypher H3K4me3 antibody shows similar results in both ChIP-seq and CUT&RUN, where specific enrichment is localized to single peaks at gene transcription start sites. In contrast, the ENCODE H3K4me3 antibody known to cross-react with H3K4me2 (Shah et al., Mol. Cell 2018) shows a localization pattern more similar to the H3K4me2 antibody. 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 in EpiCypher's CUT&RUN Protocol. Library preparation was performed with 10 ng DNA using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®.ChIP libraries were sequenced on an Illumina NextSeq 550 (2x150 bp).

Figure 4: 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.

Figure 5: 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 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 = 0.811).

Technical Information

Immunogen:
A synthetic peptide corresponding to histone H3 trimethylated at lysine 4.
Formulation:
Protein A affinity-purified antibody (0.5 mg/mL) in PBS pH 7.4 with 0.09% sodium azide.
Storage and Stability:
Stable for 1 year at -20°C from date of receipt.

Application Notes

Recommended Dilutions:

ChIP / ChIP-seq: 2 - 5 µg per 5 µg chromatin

CUT&RUN: 1:100

Luminex: 1:250 - 1:4000

References

Background References:
Grzybowski et al. Mol. Cell (2015). PMID: 26004229
Shah et al. Mol. Cell (2018). PMID: 30244833

Product References

Documents & Resources

Additional Info

Applications Key:

ChIP: Chromatin Immunoprecipitation
ChIP-seq: ChIP-sequencing
CUT&RUN: Cleavage Under Targets and Release Using Nuclease
WB: Western Blot
L: Luminex
E: ELISA
FACS: Flow Cytometry
IP: Immunoprecipitation
IF: Immunofluorescence
IHC: Immunohistochemistry
ICC: Immunocytochemistry

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
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