ChIP results obtained with the antibody directed against H3K27me2
ChIP assays were performed using human HeLa cells, the antibody against H3K27me2 (Cat. # 25241) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5, and 10 µg per ChIP experiment was analyzed. IgG (2 µg/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active GAPDH and EIF4A2 genes, used as negative controls, and for the promoter of the inactive HBB and the coding region of the inactive MYOD1 genes, used as positive controls. 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 H3K27me2 is preferably present at silent genes.

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K27me2 (Cat. # 25241). 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 1:480,000.

Cross reactivity tests using the antibody directed against H3K27me2
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K27me2 (Cat. # 25241) with peptides containing other modifications of histone H3 and H4 and the unmodified sequence. One hundred to 0.2 pmol of peptide containing the respective histone modification were spotted on a membrane. The antibody was used at a dilution of 1:50,000. Figure 3 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H3K27me2
Histone extracts (15 µg) from HeLa cells were analyzed by Western blot using the antibody against H3K27me2 (Cat. # 25241) 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.

Immunofluorescence using the antibody directed against H3K27me2
HeLa cells were stained with the antibody against H3K27me2 (Cat. # 25241) and with DAPI. Cells were fixed with 4% formaldehyde for 10 min. and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K27me2 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.

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