Chromatin was prepared from Saccharomyces cerevisiae cells according to the Abcam X-ChIP protocol. Saccharomyces cerevisiae cells were treated with MMS at 2mg/ml for 1 h. Treated and non-treated Saccharomyces cerevisiae cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab181447 (red), and 20µl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

All lanes : Anti-Histone H2A (phospho S129) antibody [EPR17588] - ChIP Grade (ab181447) at 1/5000 dilution

Lane 1 : Saccharomyces cerevisiae treated with 0.2% Methyl methanesulfonate for 1 hour lysate
Lane 2 : Untreated Saccharomyces cerevisiae whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Serially diluted ab181447 was bound to immobilised phospho (ab19828) - or control (ab19751) peptides (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.

Dot blot analysis of Histone H2A (phospho S129) peptide (Lane 1), and non-phospho peptide (Lane 2), labeled using ab181447 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time : 3 minutes

ab181447 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.