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Figure 1.ChIP results obtained with the Diagenode monoclonal antibody directed against H3K36me3 ChIP assays were performed using human HeLa cells, the Diagenode monoclonal antibody against H3K36me3 (Cat. No. C15200183) and optimized PCR primer pairs for qPCR. ChIP was performed with the “Auto Histone ChIP-seq” kit (Cat. No. C01010020), using sheared chromatin from 1 million cells. A titration consisting of 0.5, 1, 2 and 5 μg of antibody per ChIP experiment was analyzed. IgG (1 μg/IP) was used as a negative IP control. Quantitative PCR was performed with primers specific for the coding regions of the active EIF2S3 and CCT5 genes, used as positive controls, and for the inactive MYOD1 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. Cross reactivity of the Diagenode monoclonal antibody directed against H3K36me3 To test the specificity an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody against H3K36me3 (Cat. No. MAb-183-050). The wells were coated with peptides containing the unmodified H3K36 region as well as the mono-, di- and trimethylated H3K36 and the trimethylated H3K9. Figure 1 shows a high specificity of the antibody for the peptide containing the modification of interest.
Figure 3. Immunofluorescence using the Diagenode monoclonal antibody directed against H3K36me3 HeLa cells were stained with the Diagenode antibody against H3K36me3 (Cat. No. MAb-183-050) and with DAPI. Cells were fixed with methanol and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K36me3 antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
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Reader domain specificity and lysine demethylase-4 family function Su Z. et al. The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences ...