Histone

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

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

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). High target efficiency is confirmed by consistent genomic enrichment at 500k and 50k starting cells. 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) [1].

*A mixture of multiple monoclonal antibodies to ensure robust target recognition (often called "recombinant polyclonal")

Validation Data

Figure 1: SNAP specificity analysis
CUT&RUN was performed on 500k and 50k native K-562 cells with the SNAP-CUTANA™ K-MetStat Panel (EpiCypher 19-1002) spiked-in prior to the addition of 0.5 µg H3K4me3 antibody. CUT&RUN sequencing reads were aligned to the unique DNA barcodes corresponding to each nucleosome in the SNAP-CUTANA™ panel (x-axis). Data are expressed as a percent relative to on-target recovery (H3K4me3 set to 100%). Error bars represent mean ± SEM for the duplicate DNA barcodes in a single CUT&RUN experiment.

Figure 2: CUT&RUN genome wide enrichment
CUT&RUN was performed as above (Figure 1). 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 H3K4me3 - 50k 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 that was consistent between 500k and 50k cells, while IgG negative control antibody (EpiCypher 13-0042) displayed minimal background.

Figure 3: H3K4me3 CUT&RUN representative tracks at 500k and 50k cells
Gene browser shots generated using the Integrative Genomics Viewer (IGV, Broad Institute) show representative loci for H3K4me3 antibody tested in CUT&RUN using 500k and 50k cells. Robust, specific enrichment is observed at gene transcription start sites for both cell inputs. Methods: CUT&RUN was performed using the CUTANA™ ChIC/CUT&RUN Kit v2.0 (EpiCypher 14-1048) adapted to high throughput Tecan liquid handling. Library preparation was performed with 5 ng DNA using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 5.7 million reads (500k cell input) and 5.6 million reads (50k cell input).

Figure 4: CUT&RUN cell input correlation analysis
Genome-wide correlation analysis was performed to compare H3K4me3 antibody enrichment in CUT&RUN 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 = 0.828), indicating high consistency of H3K4me3 antibody target recovery.

Figure 5: Multiplexed specificity profiling
Histone H3K4me3 antibody was assessed using a Luminex® based approach employing dCypher™ Nucleosome K-MetStat Panel (EpiCypher 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 1:250 dilution and detected with a 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 6: Immunofluorescence
Representative images (60X magnification) of HeLa cells fixed, permeabilized, and immunostained to show endogenous localization of H3K4me3. Panel a: H3K4me3 antibody (green, 1:100 dilution) detected with an Alexa Fluor® 488 conjugated anti-rabbit secondary. Panel b: DAPI stained nuclei (blue). Panel c: Rhodamine stained cytoskeletal F-actin (red). Panel d: A composite of panels a, b & c demonstrating the nuclear localization of H3K4me3. Panel e: Negative control lacking H3K4me3 primary antibody to assess background.

Figure 7: Western blot data
Western analysis of H3K4me3 in whole cell extracts from HeLa, Hep G2, HCT 116, MCF7, U-2 OS, AF49, 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 8: 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 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.
Formulation
Protein A affinity-purified antibody in PBS pH 7.4, with 0.09% sodium azide.
Storage and Stability
Stable for 1 year at -20°C from date of receipt.

Recommended Dilution

CUT&RUN: 0.5 µg per reaction

Chromatin Immunoprecipitation (ChIP): 2 - 5 µg per 5 µg chromatin

Immunocytochemistry/Immunofluorescence (ICC/IF): 1:100

Western Blot (WB): 1:250

References

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

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