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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K27ac ChIP assays were performed using human HeLa cells, the Diagenode monoclonal antibody against H3K27ac (Cat. No. C15200184) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010020), using sheared chromatin from 1 million cells. A titration consisting of 0.5, 1, 2 and 5 µg of antibody per ChIP experiment was analyzed. IgG (1µg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the promoters of the EIF4A2 and GAPDH genes, used as positive controls, and for the inactive MYOD1 gene and the Sat2 satellite repeat, used as negative 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. Cross reactivity of the Diagenode monoclonal antibody directed against H3K27ac To test the specificity an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody against H3K27ac (Cat. No. C15410184). The wells were coated with peptides containing the unmodified H3K27 region as well as the acetylated H3K27 and the acetylated H3K9. Figure 2 shows a high specificity of the antibody for the peptide containing the modification of interest.
Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K27ac HeLa cells were stained with the Diagenode antibody against H3K27ac (Cat. No. C15410184) 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 H3K27ac antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
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