H3K9me3 Antibody

Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K9me3
A. ChIP was performed using HeLa cells, the monoclonal antibody against H3K9me3 (Cat. No. MAb-153-050) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode Bioruptor using the “Shearing ChIP” kit (Cat. No. kch- redmod-100). ChIP was performed with the “OneDay ChIP” kit (Cat. No. kch-oneDIP-060), using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 1, 3 and 9 μg per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoter and the coding region of the GAPDH gene, and for the RPL10 and HBB promoters.
B. ChIP was performed with the “iDeal ChIP” kit (Cat. No. AB-001-0024), using sheared chromatin from 1 million K562 cells. A titration of the antibody consisting of 1, 2 and 5 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the GAPDH and EIF4A2 genes, used as negative controls, and for the ZNF12 gene and the Sat2 satellite repeat, used as positive controls. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Figure 2. Cross reactivity of the Diagenode monoclonal antibody directed against H3K9me3
To test the specificity an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody against H3K9me3 (Cat. No. MAb-153-050). The wells were coated with peptides containing the unmodified H3K9 as well as the mono-, di- and trimethylated H3K9 and the trimethylated H3K4 and H3K27. Figure 2 shows a high specificity of the antibody for the modification of interest.

Figure 3. Western blot analysis using the Diagenode monoclonal antibody directed against H3K9me3
Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the Diagenode monoclonal antibody against H3K9me3 (Cat. No. MAb-153-050) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.

Figure 4. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K9me3
HeLa cells were stained with the Diagenode antibody against H3K9me3 (Cat. No. MAb-153-050) 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 H3K9me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.