Recombinant Anti-Histone H3 (phospho S10) antibody [EPR17246] - ChIP Grade (ab177218)

Positive staining on paraffin-embedded sections of Human tonsil. Heat-mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1:1000 dilution (2.37 μg/ml). A ready-to-use Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Hematoxylin was used as the counterstain.  A secondary antibody-only control was also perfomed.

Positive staining on paraffin-embedded sections of Human ovarian carcinoma. Heat-mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1:1000 dilution (2.37 μg/ml). A ready-to-use Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Hematoxylin was used as the counterstain.  A secondary antibody-only control was also perfomed.

Positive staining on paraffin-embedded sections of Mouse spleen. Heat-mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1:8000 dilution (0.3 μg/ml). A ready-to-use Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Hematoxylin was used as the counterstain.  A secondary antibody-only control was also perfomed.

Positive staining on paraffin-embedded sections of Rat spleen. Heat-mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at a 1:8000 dilution (0.3 μg/ml). A ready-to-use Goat Anti-Rabbit IgG H&L (HRP Polymer) was used as the secondary antibody. Hematoxylin was used as the counterstain.  A secondary antibody-only control was also perfomed.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (phospho S10) with ab177218 at 1/5000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab177218 at 1/5000 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Blocking/Dilution buffer: 5% BSA /TBST.

Blocking/Dilution buffer: 5% BSA /TBST.

Chromatin was prepared from HeLa (Human epithelial cells from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. HeLa (Human epithelial cells from cervix adenocarcinoma) cells were treated with colcemid at 150ng/ml for 20 h. Treated and non-treated cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab177218 (blue), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

ab177218 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.