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

Lane 1 : HepG2 cell lysate – treated with etoposide
Lane 2 : HepG2 cell lysate – untreated

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 15 kDa

Blocking buffer - 5% NFDM/TBST

Diluting buffer - 1% BSA

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.

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).

IHC-P image of γH2A.X staining on Rat testis sections using unpurified ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with unpurified ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

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Dot blot analysis of Histone H2A.X single phospho peptide pS139 (lane 1) and Histone H2A.X non-phospho peptide (lane 2) with ab81299 at 1/1000. Blocking and diluting buffer was 5% NFDM/TBST. The secondary antibody used was ab97051 Peroxidase conjugated Goat Anti-Rabbit IgG, (H+L) at 1/100,000.

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

Lane 1 : Jurkat cell lysate - untreated
Lane 2 : Jurkat cell lysate - treated with etoposide

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP Labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa

IHC-P image of γH2A.X staining on Mouse testis sections using unpurified ab81299 (1:5000). The sections were deparaffinized and subjected to heat mediated antigen retrieval using citric acid. The sections were blocked using 1% BSA for 10 mins at 21°C. ab81299 was diluted 1:5000 in TBS buffer (containing BSA and Azide) and sections were then incubated with unpurified ab81299 for 2 hours at 21°C. The secondary antibody used was Biotin conjugated Goat polyclonal to Rabbit IgG (1:250).

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Immunohistochemical staining of paraffin embedded human brain with purified ab81299 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

Immunohistochemical staining of paraffin embedded human brain with unpurified ab81299 at a working dilution of 1 in 50. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human kidney transitional cell carcinoma using unpurified ab81299 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab81299 staining H2A.X in Human Testis tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in Citric acid. Samples were incubated with primary antibody (1/50 in TBS) for 2 hours at 21°C. A biotin conjugated Anti-Rabbit IgG (goat polyclonal) was used as the secondary antibody at a 1/250 dilution.

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