Epicypher 13-0041,Histone H3K4me3 Antibody

EpiCypher是一家为表观遗传学和染色质生物学研究提供高质量试剂和工具的专业制造商。EpiCypher推出的CUT&RUN和ChIP级别的Histone H3K4me3 Antibody符合EpiCypher的批次特异性SNAP-CertifiedTM标准，在CUT&RUN和ChIP的应用中具有高特异性和高效的靶标富集能力。在CUT&RUN中，使用SNAP-CUTANATM K-MetStat Panel测定对照组的峰值(EpiCypher 19-1002，Fig.1)，相关组蛋白PTMs的交叉反应性<20%。在500k和50k起始细胞中一致的基因组富集证实了高靶标效率(Fig. 2-4)。在ChIP中，使用SNAP-ChIP®K-MetStat Panel测定对照组的峰值 (EpiCypher 19-1001，Fig. 7)，相关组蛋白PTMs的交叉反应性<20%，并且确定目标输入回收率>5%。

产品详情

反应种属：Human, Mouse, Drosophila, Yeast, Wide Range (Predicted)

宿主来源：Rabbit

实验应用：CUT&RUN, ChIP, ICC/IF, WB

免疫原：A synthetic peptide corresponding to histone H3 trimethylated at lysine 4

克隆性：Mixed Monoclonal*

*Mixed Monoclonal: a pool of multiple monoclonal antibodies.

保存温度：Stable for 1 year at -20°C from date of receipt

运输温度：Frozen cold packs.

产品形式：Protein A affinity-purified antibody in PBS pH 7.4, 0.09% sodium azide

数据示例

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 yaxis; log scale) and indicates percentage of the target immunoprecipitated relative to input. Error bars represent mean ± SEM in replicate ChIP experiments.

抗体使用文献引用

1. Shah et al.Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies. Mol. Cell (2018). PMID: 30244833.

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