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Figure 1. ChIP results obtained with the Diagenode monoclonal antibody directed against H3K36me3 ChIP was performed with the Diagenode antibody against H3K36me3 (cat. No. C15210013) on sheared chromatin from 500,000 HeLaS3 cells using the “iDeal ChIP-seq” kit (cat. No. C01010051). A titration of the antibody consisting of 0.5, 1, and 2 µg per ChIP experiment was analysed. IgG (1 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers specific for the CCT5 coding region, used as positive control, and for a region upstream the ACTB promoter, used as negative control. The graph 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 on sheared chromatin from 500,000 HeLaS3 cells using 1 µg of the Diagenode antibody against H3K36me3 (cat. No. C15210013) as described above. The IP'd DNA was subsequently analysed on an Illumina NovaSeq. Library preparation, cluster generation and sequencing were performed according to the manufacturer's instructions. The 51 bp tags were aligned to the human genome using the BWA algorithm. Figure 2 shows the H3K36me3 signal along the complete sequence and 1.5 Mb region of human chromosome 1 (figure 2A and B), in a genomic region on chromosome X surrounding the EIF2S3 gene (figure 2C) and in a genomic region surrounding the CCT5 positive control (figure 2D).
Figure 3. Cross reactivity tests using the Diagenode monoclonal antibody directed against H3K36me3 To test the cross reactivity of the Diagenode antibody against H3K36me3 (cat. No. C15210013), a Dot Blot analysis was performed with peptides containing other histone modifications and the unmodified H3K36. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. Figure 2 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode monoclonal antibody directed against H3K36me3 Western blot was performed on recombinant histone H3 (1 µg) and on histone extracts from HeLa cells (40 µg) using the Diagenode antibody against H3K36me3 (cat. No. C15210013). The antibody was diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is shown on the right, the marker (in kDa) is shown on the left.
WB Western blot : The quality of antibodies used in this technique is crucial for correct and specific protein identification. Diagenode offers huge selection of highly sensitive and specific western blot-validated antibodies.
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