H3K36me2 Polyclonal Antibody

ChIP assays were performed using human HeLa cells, the H3K36me2 antibody (bs-53071R) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 10,000 cells. A titration of the antibody consisting of 1, 5, and 10 \u03bcl per ChIP experiment was analyzed. Additionally, the same titration was analyzed after incubation of the antibody with 5 nmol blocking peptide for 1 hour at room temperature. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active genes GAPDH and ALDOA and for the coding region of the myogenic differentiation gene (MYOD). The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

To determine the titer, an ELISA was performed using a serial dilution of the H3K36me2 antibody (bs-53071R). The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:31,000.

Histone extracts of HeLa cells (15 \u03bcg) were analysed by Western blot using the H3K36me2 antibody (bs-53071R) 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. The result of the Western analysis with the antibody is shown in lane 1; lane 2 shows the same analysis after incubation of the antibody with 5 nmol blocking peptide for 1 hour at room temperature.

A dot blot analysis was performed to test the cross reactivity of the H3K36me2 antibody (bs-53071R) with peptides containing other modifications and unmodified sequences of histone H3. 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 1:100,000. The figure shows a high specificity of the antibody for the modification of interest.

HeLa cells were stained with the H3K36me2 antibody (bs-53071R) and with DAPI. Cells were fixed with 4% formaldehyde for 10\u2019 and blocked with PBS\/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K36me2 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.