H3K4me3 polyclonal antibody - Classic

Figure 1 ChIP results obtained with the Diagenode antibody directed against H3K4me3
ChIP assays were performed using human U2OS cells, the Diagenode antibody against H3K4me3 (Cat. No. CSP-003-050) and optimized PCR primer pairs for qPCR. ChIP was performed with the “OneDay ChIP” kit (Cat. No. kch-oneDIP-060), using sheared chromatin from 2 million cells and stringent washing conditions. A titration consisting of 0.1, 1 and 3 μl 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 myoglobin exon 2. 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 trimethylation of K4 at histone H3 is associated with the promoters of active 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 against H3K4me3 (Cat. No. CSP-003-050), the crude serum and the supernatant 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 1:5,250.

Figure 3 Cross reactivity tests using the Diagenode antibody directed against H3K4me3
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me3 (Cat. No. CSP-003-050) with peptides containing other H3K4 methylations and the unmodified sequence. One hundred to 0.2 pmol of the respective peptides 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 H3K4me3
Histone extracts from HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K4me3 (Cat. No. CSP-003-050) diluted 1:250 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.