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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K9un ChIP assays were performed using human HeLa cells, the Diagenode monclonal antibody against H3K9un (Cat. No. MAb-187-050) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. AB-Auto02-A100) on sheared chromatin from 1 million cells. using the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (2 μg/IP) was used as negative IP control. QPCR was performed with primers for the coding regions of the MYOD1 and MB genes and for a region 1 kb upstream of the GAPDH promoter, used as positive controls, and for the ZNF510 coding region, the EIF4A2 promoter and the Sat2 satellite repeat region, 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).
Figure 2. Western blot analysis using the Diagenode monoclonal antibody directed against H3K9un Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K9un (Cat. No. MAb-187-050). The antibody was 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 3. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K9un HeLa cells were stained with the Diagenode antibody against H3K9un (cat. No. MAb-187-050) 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 H3K9un 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.
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