H3K9acS10p Antibody

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
HeLa cells were stained with the Diagenode antibody against H3K9acS10p (cat. No. C15310102) 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 labelled with the H3K9acS10p 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.