Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y](AB81299)

Immunofluorescence staining of A549 (Human lung carcinoma cell line) labeling gamma H2A.X (phospho S139) (green) with ab81299.

Cells were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in PBS-0.2% Triton for 10 minutes. After blocked for 1 hour, primary antibody was diluted in blocking buffer (1/100) and incubated with fixed cells overnight at 4°C. Cells were washed and incubated with secondary antibodies (1/100) for 1 hour at room temperature. All slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence was performed using confocal laser scanning microscopy (Lecia) or fluorescence microscopy (Olympus).

Dexamethasone, DEX; Cisplatin, DDP.

ab81299 at 1/40 immunoprecipitating Histone H2A.X (phospho S139) in HepG2 (human hepatocellular carcinoma epithelial) whole cell lysate observed at 15 KDa (lanes 1 and 2).

Lane 1 (input): HepG2 treated with etoposide and TSA whole cell lysate 10μg

Lane 2 (+): ab81299 + HepG2 treated with etoposide and TSA whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab81299 in HepG2 treated with etoposide and TSA

For western blotting, ab81299 (Purified) at 1/200 dilution and VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299)

Predicted band size: 15 kDa

Exposure time: 30s

Blocking buffer - 5% NFDM/TBST

Diluting buffer - 1% BSA

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/100000 dilution

Lane 1:

HepG2 cell lysate – treated with etoposide at 20 µg

Lane 2:

HepG2 cell lysate – untreated at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 15 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with H2O2) labelling Histone H2A.X (phospho S139) with ab81299 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei counterstained with DAPI (blue).

Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (ab81299) at 1/500000 dilution

Lane 1:

Jurkat cell lysate - untreated at 10 µg

Lane 2:

Jurkat cell lysate - treated with etoposide at 10 µg

Secondary

All lanes:

HRP Labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa