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Figure 1. ChIP results obtained with the Diagenode antibody directed against H2AK119ub. ChIP assays were performed using human HeLa cells, the Diagenode antibody against H2AK119ub (cat. No. C15410002) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (cat. No. AB-Auto02-A100), using sheared chromatin from 1 million cells on the SX-8G IP-Star automated system. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the active GAPDH and c-fos genes, used as negative controls, and for the inactive MYT1 and TSH2B genes, used as a 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 antibody titer. To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody against H2AK119ub (cat. No. C15410002). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:23,000.
Figure 3. Cross reactivity tests using the Diagenode antibody directed against H2AK119ub To test the cross reactivity of the Diagenode antibody against H2AK119ub (cat. No. C15410002), a Dot Blot analysis was performed with peptides containing other histone ubiquitinylations and unmodified sequences. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode antibody directed against H2AK119ub. Western blot was performed on whole cell extracts from HeLa cells (25 μg, lane 1), and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 2, 3, 4 and 5, respectively) using the Diagenode antibody against H2AK119ub (cat. No. C15410002). The antibody was diluted 1:200 in TBS- Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.
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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