H3K27me3 monoclonal antibody

Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K27me3
ChIP was performed with the Diagenode antibody against H3K27me3 (cat. No. C15210017) on sheared chromatin from 500,000 HeLaS3 cells using the “iDeal ChIP-seq” kit (cat. No. C01010051). A titration of the antibody consisting of 0.5, 1, and 2 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the TSH2B and MYT1 genes, used as positive controls, and for the promoters of the GAPDH and EIF4A2 genes, used as negative controls. The graph shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K27me3
ChIP was performed on sheared chromatin from 500,000 HeLaS3 cells using 0.5 µg of the Diagenode antibody against H3K27me3 (cat. No. C15210017) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the signal distribution along the complete sequence of chromosome 1 and a zoomin to a 3 Mb region (fig 2A and B) and in two genomic regions surrounding the MYT1 and TSH2B positive control genes (fig 2C and D, respectively).

Figure 3. Cross reactivity tests using the Diagenode monoclonal antibody directed against H3K27me3
To test the cross reactivity of the Diagenode antibody against H3K27me3 (cat. No. C15210017), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K27. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 2 shows a high specificity of the antibody for the modification of interest.

Figure 4. Western blot analysis using the Diagenode monoclonal antibody directed against H3K27me3
Western blot was performed on whole cell extracts (40 µg) from HeLa cells using the Diagenode antibody against H3K27me3 (cat. No. C15210017). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.