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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9acS10p ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against H3K9acS10p (Cat. No. CS-102-100) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode “Shearing ChIP” kit (Cat. No. kch-redmod-100). ChIP was performed with the “OneDay ChIP” kit (Cat. No. kch-oneDIP-060), using sheared chromatin from 1.5 million cells. A titration of the antibody consisting of 5 and 15 μl per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the ALDOA (fructose-bisphosphate aldolase A) promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. 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, an ELISA was performed using a serial dilution of the Diagenode antibody against human H3K9acS10p (Cat. No. CS-102-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:89,000.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K9acS10p A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9acS10p (Cat. No. CS-102-100) with peptides containing other modifications of histone H4 and H3 or unmodified histone H3 sequences. One hundred to 0.2 pmol of the peptide containing the respective histone modification 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 double modification.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3K9acS10p Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K9acS10p (Cat. No. CS-102-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right.
Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K9acS10p Figure 5. Immunofluorescence using the Diagenode antibody directed against H3K9acS10p
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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