Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K36me3
ChIP assays were performed using HeLa cells, the Diagenode antibody against H3K36me3 (cat. No. C15410058) and optimized PCR primer sets for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (cat. No. C01010022), using sheared chromatin from 1 million cells on the SX-8G IP-Star automated system. 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. QPCR was performed with primers for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter (cat. No. C17011003) and for the Sat2 satellite repeat. 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 H3K36me3
ChIP was performed with 2 µg of the Diagenode antibody against H3K36me3 (cat. No. C15410058) on sheared chromatin from 1 million HeLaS3 cells using the “iDeal ChIP-seq” kit (cat. No. C01010051). IgG (2 µg/IP) was used as a negative IP control. The IP'd DNA was analysed by QPCR with optimized PCR primer pairs for the promoter and coding region of the active GAPDH, for a region located 1 kb upstream of the GAPDH promoter and for the coding region of the active ACTB gene (figure 2A). 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 2B shows the obtained profiles in genomic regions of chromosome 12 (including the GAPDH positive control), 7 (including the ACTB positive control), 14 and 3, respectively. These results clearly show an enrichment of the H3K36me3 at active genes.

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 H3K36me3 (cat. No. C15410058) and the crude serum. 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 purified antibody was estimated to be 1:19,300.

Figure 4. Cross reactivity tests using the Diagenode antibody directed against H3K36me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K36me3 (cat. No. C15410058) with peptides containing other H3 and H4 modifications and the unmodified sequence. 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 1:20,000. 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 H3K36me3
Histone extracts (15 µg) from HeLa cells were analysed by Western blot using the Diagenode antibody directed against H3K36me3 (cat. No. C15410058) diluted 1:1,000 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.