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Figure 1 ChIP results obtained with the Diagenode antibody directed against H2B ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2B (cat. No. C15410157) 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 0.5, 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 active GAPDH and EIF4A2 genes, 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 H2B (C15410157) in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:40,000.
Figure 3 Western blot analysis using the Diagenode antibody directed against H2B Western blot was performed on whole cell extracts from HeLa cells (40 µg, lane 1) and on 1 µg of recombinant histone H2B (lane 2) using the Diagenode antibody against H2B (Cat. No. C15410157). The antibody was diluted 1:10,000 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 H2B HeLa cells were stained with the Diagenode antibody against H2B (Cat. No. C15410157) 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 H2B 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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