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Figure 1 ChIP results obtained with the Diagenode antibody directed against H3K36me1 ChIP assays were performed using human U2OS cells, the Diagenode antibody against H3K36me1 (cat. No. pAb-089-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “OneDay ChIP” kit (cat. No. kch-oneDIP-060), using sheared chromatin from 1.5 million cells and stringent washing conditions. A titration consisting of 1, 2, 5 and 10 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 for the promoter of the constitutively expressed GAPDH gene 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).
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 H3K36me1 (cat. No. pAb-089-050), crude serum and Flow Through. 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 1:46,000.
Figure 3 Cross reactivity tests using the Diagenode antibody directed against H3K36me1 A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K36me1 (cat. No. pAb-089-050) with peptides containing other modifications of histone H3 or the unmodified H3K36. 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 H3K36me1 Histone extracts (15 μg) from HeLa cells were analysed by Western blot using the Diagenode antibody against H3K36me1 (cat. No. pAb-089-050) 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.
Figure 5 Immunofluorescence using the Diagenode antibody directed against H3K36me1 Human osteosarcoma (U2OS) cells were stained with the Diagenode antibody against H3K36me1 (cat. No. pAb- 089-050) and with DAPI. Cells were fixed with 4% formaldehyde for 20’ and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 5A: cells were immunofluorescently labeled with the H3K36me1 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure 5B, C, D and E: staining of the cells with the H3K36me1 antibody after incubation of the antibody with 2 ng/μl blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K36, respectively.
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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