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Figure 1. ChIP results obtained with the Diagenode antibody directed against H2Apan ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2Apan (cat. No. C15410166) and optimized PCR primer sets for qPCR. ChIP was performed with the Auto Histone ChIP-seq” kit (cat. No. C01010022) on sheared chromatin from 1 million cells using the IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 μg per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH and EIF4A2 promoters, used as negative controls and for the inactive MYOD1 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. Determination of the titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H2Apan (cat. No. C15410166) in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:32,500.
Figure 3. Western blot analysis using the Diagenode antibody directed against H2Apan 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 H2Apan (cat. No. C15410166). The antibody was diluted 1:2,000 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 antibody directed against H2Apan HeLa cells were stained with the Diagenode antibody against H2Apan (Cat. C15410166) 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 labeled with the H2Apan 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.
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