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Figure 1. ChIP-seq results obtained with the Diagenode antibody directed against H3K36me3 ChIP was performed with 5 µl of the Diagenode antibody against H3K36me3 (cat. No. CS-058-050) on sheared chromatin from 1 million HeLaS3 cells using the “Auto Histone ChIP-seq” kit (cat. No. AB-Auto02-A100) on the IP-Star automated system. 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 coding and promoter region of the active GAPDH gene, for the coding region of the inactive TSH2B gene and for the Sat2 satellite repeat (figure 2A). The IP’d DNA was subsequently analysed with 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 results in 200 kb regions of chromosome 12 (including the GAPDH positive control), 6 and 7 and 14. These results clearly show an enrichment of the H3K36me3 at active genes
Figure 2. ChIP results obtained with the Diagenode antibody directed against H3K36me3 ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against H3K36me3 (cat. No. CS-058-100) 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. 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 a negative IP control. Quantitative PCR was performed using primer sets for the housekeeping gene GAPDH and for myogenic differentiation gene (MYOD). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis.
Figure 3. Determination of the titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K36me3 (cat. No. CS-058-100). 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 1:12,700.
Figure 4. Cross reactivity test 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. CS-058-050) with peptides containing other modifications of histone H3. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di- and trimethylation of lysine 9, 27 and 79. 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 1:100,000. 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 against H3K36me3 (cat. No. CS-058-100) diluted 1:1,000 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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