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Figure 1 ChIP results obtained with the Diagenode antibody directed against H4 ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4 (Cat. No. C15410156) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010022), using sheared chromatin from 1 million cells on the IP-Star Compact automated system. A titration consisting of 0.5, 1, 2 and 5 μg of antibody per ChIP experiment was analyzed. IgG (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoters of the active GAPDH and c-fos genes, and for the inactive MYOD1 gene and the Sat2 satellite repeat. 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 H4 (Cat. No. C15410156) in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:3,000.
Figure 3 Western blot analysis using the Diagenode antibody directed against H4 Western blot was performed on whole cell extracts (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 H4 (Cat. No. C15410156). 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 4. Immunofluorescence using the Diagenode antibody directed against H4 HeLa cells were stained with the Diagenode antibody against H4 (Cat. No. C15410156) and with DAPI. Cells were fixed with 4% formaldehyde for 10’ and blocked with PBS/TX-100 containing 1% BSA. The cells were immunofluorescently labeled with the H4 antibody (middle) diluted 1:1,000 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The left panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
WB Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.
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