Anti-gamma H2A.X (phospho S139) antibody

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2AX (phospho S139) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with camptothecin (ab120115), by ICC/IF. Increased nuclear expression of γH2AX (phospho S139) correlates with increased concentration of camptothecin, as described in literature.
The cells were incubated at 37°C for 3h in media containing different concentrations of ab120115 (camptothecin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• FuncS

Unknown

Functional Studies - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were incubated at 37°C for 3h with vehicle control (0 μM) and different concentrations of camptothecin (ab120115). Increased expression of γH2A.X (phospho S139) in HeLa cells correlates with an increase in camptothecin concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab2893 at 1 μg/ml and ab10475 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP) performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm.

Histone gamma H2A.X was detected using ab2893.

Garcia, C.P. et al PLoS One. 2016; 11(3): e0152607. Fig.2a Published online 2016 Mar 31. doi: 10.1371/journal.pone.0152607 Reproduced under the Creative Commons licence https://creativecommons.org/licenses/by/4.0/.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with SN 38 (ab141108), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of SN 38, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141108 (SN 38) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 10-Hydroxycamptothecin (ab141071), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of 10-Hydroxycamptothecin, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141071 (10-Hydroxycamptothecin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining gamma H2A.X (phospho S139) in HeLa UV cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab2893 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

Asynchronous HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were exposed to 2Gy and permitted to recover for 30min. Cells were paraformaldehyde fixed (4%), immunofluorescently labeled with ab2893 and counterstained with DAPI. The merge image presents the DAPI and ab2893 channels as red and green, respectively. The scale bar represents 5μm.

Image courtesy of Dr. Kirk McManus

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

Asynchronous HeLa cells were paraformaldehyde fixed and immunofluorescently labeled with ab2893 that had been preincubated with either 1) non-phosphorylated or 2) phosphorylated H2AX peptide. Identical exposure times were employed. The Merge images present the DAPI and ab2893 channels as red and green, respectively. Scale bars represent 5μm.

1) Non-phosphorylated peptides

2) Phosphorylated peptides

This image is courtesy of Kirk McManus

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with CPT 11 (Irinotecan) (ab141107), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of CPT 11 (Irinotecan), as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141107 (CPT 11 (Irinotecan)) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight^® 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2A.X in weri cells treated with TMPyP4 tosylate (ab120793), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of TMPyP4 tosylate, as described in literature.
The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120793 (TMPyP4 tosylate ) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

ab2893 staining γH2A.X in MALME-3M cells treated with terfenadine (ab120270), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of terfenadine, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab120270 (terfenadine) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

Gel type : MES Blocking buffer : 2% BSA block

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1 µg/mL

Lane 1:

NIH/3T3 (mouse embryonic fibroblast cell line) nuclear lysate (triton enriched) at 10 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma cell) nuclear lysate (triton enriched) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

false

Exposure time: 16min

• WB

Lab

Western blot - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

Gel type : MES

Blocking buffer : 2% BSA block

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1 µg/mL

Lane 1:

NIH 3T3 nuclear lysate (triton enriched) at 10 µg

Lane 2:

PC12 nuclear lysate (triton enriched) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

false

Exposure time: 4min

• WB

Unknown

Western blot - Anti-gamma H2A.X (phospho S139) antibody (AB2893)

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1/500 dilution

Lane 1:

Control HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate Histone preparation

Lane 2:

Colcemid treated HeLa whole cell lysate Histone preparation

Lane 3:

Control HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate Histone preparation with Human gamma H2A.X (phospho S139) peptide (<a href='/ko/products/proteins-peptides/human-gamma-h2ax-phospho-s139-peptide-ab15645'>ab15645</a>)

Lane 4:

Colcemid treated HeLa whole cell lysate Histone preparation with Human gamma H2A.X (phospho S139) peptide (<a href='/ko/products/proteins-peptides/human-gamma-h2ax-phospho-s139-peptide-ab15645'>ab15645</a>)

Lane 5:

Control HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate Histone preparation with Human Histone H2A.X (unmodified ) peptide (<a href='/ko/products/unavailable/human-histone-h2ax-unmodified-peptide-ab15646'>ab15646</a>)

Lane 6:

Colcemid treated HeLa whole cell lysate Histone preparation with Human Histone H2A.X (unmodified ) peptide (<a href='/ko/products/unavailable/human-histone-h2ax-unmodified-peptide-ab15646'>ab15646</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa,50 kDa

false