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Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K27me3S28p ChIP assays were performed using HeLa cells treated with colcemid, the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and optimized PCR primer pairs for qPCR. ChIP was performed with the “LowCell# ChIP” kit (cat. No. kch-maglow-016), using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5 and 10 μl per ChIP experiment was analysed. Additionally, ChIP was performed after incubation of the antibody with 5 nmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature. IgG (5 μg/IP) was used as negative IP control. QPCR was performed with primers for the promoters of the active genes c-fos (cat. No. pp-1004-050) and RPL30 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 titer To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K27me3S28p (cat. No. CS-091-100). The antigen used was a peptide containing the histone modifications of interest. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:8,300.
Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K27me3S28p A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) with peptides containing other modifications of histone H3 and H4 and unmodified sequences from histone H3. One hundred to 0.2 pmol of the peptides 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 peptide containing the modifications of interest.
Figure 4. Western blot analysis using the Diagenode antibody directed against H3K27me3S28p Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) diluted 1:250 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right. Lane 2 shows the result of the Western analysis with the antibody; lane 1 shows the same analysis after incubation of the antibody with 750 pmol blocking peptide (cat. No. sp-091-050) for 1 hour at room temperature.
Figure 5. Immunofluorescence with the Diagenode antibody directed against H3K27me3S28p Hela asynchronous cells were stained with the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100) and with DAPI. Cells were fixed with formaldehyde, permeabilized with sodium citrate and Triton X100 and blocked with PBS containing 2.5% BSA. Figure 5A: cells were immunofluorescently labelled with the H3K27me3S28p antibody (diluted 1:200 and incubated for 1 hour at room temperature) followed by goat anti-rabbit antibody conjugated to DyLight 488. Figure 5B: the nuclei were stained with DAPI, which specifically labels DNA. Phosphorylation of H3 on serine 28 is increased during late G2 phase and reaches a maximum in metaphase cells. This may explain the different staining intensities of different cells.
Figure 6. Immunoprecipitation with the Diagenode antibody directed against H3K27me3S28p HeLa cells were treated with colcemid to block the cell cycle in metaphase and were fixed with formaldehyde. Chromatin from 10,000 cells was sheared and used for immunoprecipitation (IP). IP was performed with 5 μl of the Diagenode antibody against H3K27me3S28p (cat. No. CS-091-100). The immunoprecipitated proteins were analysed by Western blot with the antibody diluted 1:500 in TBS-Tween containing 5% skimmed milk. Lane 1 shows the result of the IP; a negative IP control (no antibody added) and a positive control (sheared chromatin from 10,000 cells) are shown in lane 2 and 3, respectively.
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