ChIP results obtained with the antibody directed against H3K79me3
ChIP was performed with the antibody against H3K79me3 (Cat. # 25263) on sheared chromatin from 1 million HeLaS3 cells. A titration of the antibody consisting of 1, 2, 5 and 10 µg per ChIP experiment was analyzed. IgG (5 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for exon 2 of the inactive myoglobin gene. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K79me3 shows a preference for active promoters. 

ChIP-seq results obtained with the antibody directed against H3K79me3
ChIP was performed as described above using 2 µg of the antibody against H3K79me3 (Cat. # 25263). The immunoprecipitated DNA was analyzed 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 600 kb region of the X-chromosome (figure 2A and B), in a 2 Mb region from chromosome 11 (figure 2C), and in a 100 kb region surrounding the GAPDH positive control gene (figure 2D). 

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of antibody directed against H3K79me3 (Cat. # 25263), crude serum and flow-through in antigen coated wells. 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 1:3,500.

Cross reactivity tests using the antibody directed against H3K79me3
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K79me3 (Cat. # 25263) 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.

Western blot analysis using the antibody directed against H3K79me3
Histone extracts of HeLa cells (15 µg) were analyzed by Western blot using the antibody against H3K79me3 (Cat. # 25263), diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The molecular weight marker is shown on the right; the location of the protein of interest is indicated on the left. 

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