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Figure 1 ChIP results obtained with the Diagenode antibody directed against H3K9me1 ChIP assays were performed using human HeLa cells, the Diagenode antibody directed against H3K9me1 (Cat. No. pAb-065-050) and optimized PCR primer sets for qPCR. ChIP was performed with the “LowCell# ChIP” kit (Cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. Respectively 1 and 5 μg of the antibody and 5 μg of IgG (negative IP control) were used per ChIP experiment. QPCR was performed with primers for the GAPDH promoter and for the inactive 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). These results are in accordance with the observation that H3K9me1 is preferably present at silent genes.
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 human H3K9me1 (Cat. No. pAb-065-050), crude serum and flow through in antigen coated wells. 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 1:68,000.
Figure 3 Cross reactivity test of the Diagenode antibody directed against H3K9me1 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K9me1 (Cat. No. pAb-065-050) with peptides containing other modifications and the unmodified sequence of histone H3. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:20,000. 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 H3K9me1 Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the Diagenode antibody against H3K9me1 (Cat. No. pAb-065-050) 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.
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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The histone demethylase enzyme KDM3A is a key estrogen receptor regulator in breast cancer. Wade MA, Jones D, Wilson L, Stockley J, Coffey K, Robson CN, Gaughan L Endocrine therapy has successfully been used to treat estrogen receptor (ER)-positive breast cancer, but this invariably fails with cancers becoming refractory to treatment. Emerging evidence has suggested that fluctuations in ER co-regulatory protein expression may facilitate resistance to therapy and be involved i...