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Synthetic peptide corresponding to Saccharomyces cerevisiae Histone H2A aa 100 to the C-terminus (phospho S129) conjugated to Bovine Serum Albumin (BSA).
Database link: P04911
Our Abpromise guarantee covers the use of ab15083 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/500 - 1/1000. Detects a band of approximately 14 kDa (predicted molecular weight: 14 kDa).|
|ChIP||Use at an assay dependent concentration.|
|PepArr||Use a concentration of 0.2 - 0.02 µg/ml.|
The blots were produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membranes were then blocked for an hour. 1 microgram per mL of control- (ab19751, lane 1 and 2) or phospho-peptides (ab19828, lane 3 and 4) were added to the primary antibody ab15083 (rabbit anti-Histone H2A (phospho S129) antibody; diluted 1:500) and loading control ab125247 (mouse anti-GAPDH antibody; diluted 1:20000) and the membranes were incubated with peptide/antibody mixture for 24 hours at 4°C. Antibody binding was detected using infrared (IR)-labelled goat anti-rabbit (green) and IR-labelled goat anti-mouse (red; insert below) antibodies, diluted 1:20,000, for 1 hour at room temperature before imaging.
All batches of ab15083 are tested in Peptide Array against peptides to different Histone H2A modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H2A - phospho S129 (ab19828), indicating that this antibody specifically recognises the Histone H2A - phospho S129 modification.
ab19828 - Histone H2A - phospho S129
ab19751 - Histone H2A - unmodified
Whole cell extracts from wild-type yeast strains were analyzed by 15% SDS-PAGE followed by Western botting using either the affinity purified anti-P-S129 H2A (ab15083) or anti-H2A for a loading control. The MMS treated ells were grown in the presence of 0.10% MMS for 30 min at 30°C prior to lysing.
Yeast proteins were prepared by TCA precipitation of whole cell extracts from wild-type yeast in mid-log phase, either treated (lane 1) or not treated (lane 2) with 0.2% MMS for one hr prior to harvesting.
Extracts were electrophorised on 18% SDS PAGE, transferred to nitrocellulose and blocked for 1 hr in 5% Marvel in TBS-T. The membrane was incubated with ab15083 at 1:1000 in TBS-T for 1 hr at RT, washed and incubated with anti-rabbit secondary (Pierce) for 1 hr at 1:10,000in TBS-T for 1 hr at RT. The blots were visualised with Pierce ECL.