H3K4me2 polyclonal antibody

Figure 1. ChIP results obtained with the Diagenode antibody directed against H3K4me2
ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against H3K4me2 (cat. No. CS-035-100) and optimized PCR primer sets for qPCR. Chromatin was sheared with the Diagenode “Shearing ChIP” kit (cat. No. kch-redmod-100). ChIP was performed with the “OneDay ChIP” kit (cat. No. kch-oneDIP-060), using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 2, 5, 10 or 15 μl per ChIP experiment was analysed. IgG (5 μg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoter of the ALDOA gene and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. 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 dimethylation of K4 at histone H3 is more present at active genes than at silent genes.

Figure 2. Determination of the titer
To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against H3K4me2 (cat. No. CS-035-100). 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 1:2,600.

Figure 3. Cross reactivity test using the Diagenode antibody directed against H3K4me2
A Dot Blot analysis was performed to test the cross reactivity of the Diagenode antibody against H3K4me2 (cat. No. CS-035-100) with peptides containing other modifications or unmodified sequences of histone H3. Other histone modifications include mono- and trimethylation of the same lysine and mono-, di- and trimethylation of lysine 9, 27 and 36 and 79. 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 modification of interest.

Figure 4. Western blot analysis using the Diagenode antibody directed against H3K4me2
Histone extracts of HeLa cells (15 μg) were analysed by Western blot using the Diagenode antibody against H3K4me2 (cat. No. CS-035-100) diluted 1:750 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.