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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K79me2 ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K79me2 (Cat. No. C15410051) 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 (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the coding regions of the active EIF2S3 and CCT5 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. ChIP-seq results obtained with the Diagenode antibody directed against H3K79me2 ChIP was performed with 1 μg of the Diagenode antibody against H3K79me2 (Cat. No. C15410051) as described above. The IP’d DNA was subsequently analysed on an Illumina HiSeq 2000. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the peak distribution along the complete sequence and a 5 Mb region of chromosome 1 (figure 2A and B) and in two 300 kb regions surrounding the EIF2S3 and CCT5 positive control genes (figure 2C and D).
Figure 3. Determination of the antibody titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K79me2 (Cat. No. C15410051). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 3), the titer of the antibody was estimated to be 1:6,600.
Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K79me2 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K79me2 (Cat. No. C15410051) with peptides containing other modifications and unmodified sequences of histone H3. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:5,000. Figure 4 shows a high specificity of the antibody for the modification of interest.
Figure 5. Western blot analysis using the Diagenode antibody directed against H3K79me2 Western blot was performed on whole cell (25 μg, lane 1) and histone extracts (15 μg, lane 2) from HeLa cells, and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the Diagenode antibody against H3K79me2 (Cat. No. C15410051) diluted 1:200 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.
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