H3K4me3 polyclonal antibody - Classic

Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me3
ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K4me3 (Cat. No. pAb-030-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (Cat. No. AB-001-0024), using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoter of the active genes GAPDH and EIF4A2, used as positive controls, and for exon 2 of the inactive myoglobin (MB) gene and the Sat2 satellite repeat, used as negative controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that trimethylation of K4 at histone H3 is associated with the promoters of active genes

Figure 2. ChIP-seq results obtained with the Diagenode antibody directed against H3K4me3
ChIP was performed on sheared chromatin from 1 million HeLa cells using 1 μg of the Diagenode antibody against H3K4me3 (Cat. No. pAb-030-050) as described above. The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2 shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure 2A and B) and in two regions surrounding the GAPDH and EIF4A2 positive control genes, respectively (figure 2C and D). These results clearly show an enrichment of the H3K4 trimethylation at the promoters of active genes.

Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K4me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me3 (Cat. No. pAb-030-050) with peptides containing other modifications and unmodified sequences of histone H3 and H4. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

Figure 4. Western blot analysis using the Diagenode antibody directed against H3K4me3
Western blot was performed using histone extracts from HeLa cells (HeLa HE, 15 μg) and the Diagenode antibody against H3K4me3 (Cat. No. pAb-030-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. A molecular weight marker (in kDa) is shown on the left, the position of the protein of interest is shown on the right.

Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K4me3
HeLa cells were stained with the Diagenode antibody against H3K4me3 (Cat. No. pAb-030-050) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K4me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.