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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3R17me2(asym) ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against H3R17me2(asym) (cat. No. CS-092-100) 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 1.6 million cells per ChIP reaction. A titration of the antibody consisting of 2, 5, 10 and 15 μl per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). Figure 1A: QPCR performed with primers for the GAPDH promoter (cat. No. pp-1001-050) and for exon 2 of the myoglobin gene (cat. No. pp-1006-050). Figure 1B: QPCR performed with primers for the promoter of the active ALDOA gene and for the coding region of the inactive MYOD gene..
Figure 2. Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3R17me2(asym) (cat. No. CS-092-100). 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 1:40,000.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H3R17me2(asym) A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3R17me2(asym) (cat. No. CS-092-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. 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 modification of interest.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3R17me2(asym) Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3R17me2(asym) (cat. No. CS-092-100) 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.
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Assaying epigenome functions of PRMTs and their substrates. Rakow S, Pullamsetti SS, Bauer UM, Bouchard C Among the widespread and increasing number of identified post-translational modifications (PTMs), arginine methylation is catalyzed by the protein arginine methyltransferases (PRMTs) and regulates fundamental processes in cells, such as gene regulation, RNA processing, translation and signal transduction. As epigene...