Anti-Histone H2A (phospho S129) antibody

• ELISA

Lab

ELISA - Anti-Histone H2A (phospho S129) antibody (AB15083)

Serially diluted ab15083 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.

• WB

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Western blot - Anti-Histone H2A (phospho S129) antibody (AB15083)

All lanes:

Western blot - Anti-Histone H2A (phospho S129) antibody (ab15083) at 1 µg/mL

All lanes:

S.cerevisiae (Y190) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

true

Exposure time: 90s

• WB

Lab

Western blot - Anti-Histone H2A (phospho S129) antibody (AB15083)

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 lanes:

Western blot - Anti-Histone H2A (phospho S129) antibody (ab15083) at 1/500 dilution

Lane 1:

S.cerevisiae yeast extract (SCYE) + control peptide at 10 µg with Human Histone H2A peptide (<a href='/ko/products/unavailable/human-histone-h2a-peptide-ab19751'>ab19751</a>)

Lane 2:

S.cerevisiae yeast extract with 0.2 % Methyl methanesulfonate (1 hour) at 10 µg with Human Histone H2A peptide (<a href='/ko/products/unavailable/human-histone-h2a-peptide-ab19751'>ab19751</a>)

Lane 3:

SCYE + phospho peptide at 10 µg with Histone H2A peptide - phospho S129 (ab19828)

Lane 4:

SCYE_M + phospho peptide at 10 µg with Histone H2A peptide - phospho S129 (ab19828)

Predicted band size: 14 kDa

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• PepArr

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Peptide Array - Anti-Histone H2A (phospho S129) antibody (AB15083)

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