H3S10p Polyclonal Antibody

HeLa cells were treated with colcemid which blocks the cell cycle in metaphase and 15 \u03bcg of histone extracts of the cells were analyzed by Western blot using the Bioss antibody against H3S10p (Cat. No. bs-53135R) diluted 1:1,000 in TBS-Tween containing 5% skimmed milk.

To determine the titer, an ELISA was performed using a serial dilution of the Bioss antibody directed against H3S10p (Cat. No. bs-53135R) and the crude serum. 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 1:5,200.

Figure A: ChIP assays were performed using HeLa cells treated with colcemid to block the cells in metaphase, H3S10p Polyclonal Antibody (bs-53135R) and optimized PCR primer sets for qPCR. ChIP was performed with LowCell ChIP kit on sheared chromatin from 10,000 cells using the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5, and 10 \u03bcg per ChIP experiment was analyzed. IgG (5 \u03bcg\/IP) was used as negative IP control. QPCR was performed with primers for the promoter of the active genes c-fos and RPL30. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). Figure B: ChIP was performed as described above using 2 \u03bcg of H3S10p antibody and sheared chromatin from 10,000 HeLa cells treated with colcemid (sample 1) or from 10,000 untreated cells (sample 2). QPCR was performed with primers for the promoter of the active genes c-fos and RPL30, and for the Sat2 satellite repeat region.

Human osteosarcoma (U2OS) cells were stained with H3S10p Polyclonal Antibody (bs-53135R) and with DAPI. Cells were fixed with 3.7% formaldehyde in PBS for 20\u2019 at RT, followed by a 20\u2019 permeabilization with 0.5% Triton X-100 in PBS and blocked PBS\/TX-100 containing 5% normal goat serum Figure A: cells were immunofluorescently labeled with the H3S10p antibody (left) diluted 1:2,000 in blocking buffer followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right). Figure B, C and D: staining of the cells with the H3S10p antibody after incubation of the antibody with 2 \u03bcM blocking peptide containing the unmodified H3S10 sequence, the phosphorylated H3S10, and the phosphorylated H3T11, respectively.