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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K9me3 ChIP assays were performed using undifferentiated human teratocarcinoma cells (NCCIT), the Diagenode antibody against H3K9me3 (cat. No. CS-056-100) and optimized PCR primer sets for qPCR. Sheared chromatin from 10,000 cells was used per ChIP experiment. The antibody was diluted 1:5000. The pre-immune serum (PI, diluted 1:5000) was used as a negative control. Quantitative PCR was performed using primer sets for the satellite repeat Sat2 as a positive control and for the promoter of the house keeping gene c-fos, as a negative control. 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 H3K9me3 is preferably present at heterochromatin.
Figure 2. 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 H3K9me3 (cat. No. CS-056-100). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:35,000.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K9me3 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9me3 (cat. No. CS-056-100) with peptides containing other histone modifications of histone H3 and H4. Other histone modifications include mono- and dimethylation of the same lysine and mono-, di- and trimethylation of lysine 27 and 36 of H3, and of lysine 20 of H4. 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 1:10,000. Figure 3 shows a high specificity of the antibody for the modification of interest.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3K9me3 Histone (acid) extracts of NB4 (human promyelocytic leukemia) cells were analysed by Western blot using the Diagenode antibody against H3K9me3 (cat. No. CS-056-100) diluted 1:750 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest of interest is indicated on the left.
Figure 5. Immunofluorescence analysis using the Diagenode antibody directed against H3K9me3 NIH3T3 cells (mouse fibroblasts) were stained with the antibody against H3K9me3 (cat. No. CS-056-100) and with DAPI. Cells were formaldehyde fixed, permeabilized with TritonX100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K9me3 antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to FITC. Figure 5B: staining of the nuclei with DAPI, which specifically labels DNA. Both antibody and DAPI staining are restricted to the nucleus. The dense signals obtained with both stainings characterize the distribution pattern of H3K9me3, which is linked to the transcriptionally inactive, condensed pericentric heterochromatin.
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Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9
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H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1. Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 ...