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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K79me1 ChIP assays were performed using human HeLa cells, the Diagenode antibody against H3K79me1 (Cat. No. pAb-082-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “iDeal ChIP-seq” kit (cat. No. AB-001-0024), using sheared chromatin from 1 million cells. A titration consisting of 1, 2, 5 and 10 μg of antibody per ChIP experiment was analyzed. IgG (2 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for a region 1 kb upstream of the promoter and the coding region of the active GAPDH gene, used as positive controls, and for exon 2 of the inactive myoglobin (MB) 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 H3K79me1 ChIP was performed with 1 μg of the Diagenode antibody against H3K79me1 (Cat. No. pAb-082-050) as described above and the IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure 2 shows the peak distribution along the complete sequence and a 1 Mb region of the X-chromosome (figure 2A and B), in 100 kb regions surrounding the GAPDH positive control and EIF4A2 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 H3K79me1 (Cat. No. pAb-082-050). 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 1:11,500.
Figure 4. Cross reactivity test using the Diagenode antibody directed against H3K79me1 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K79me1 (Cat. No. pAb-082-050) with peptides containing other histone modifications and the unmodified H3K79. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000. 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 H3K79me1 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 H3 (lane 3) using the Diagenode antibody against H3K79me1 (Cat. No. pAb-082-050). The antibody was 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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