Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) treated with UV-C (50 mJ/cm2) then recovery (1h) (Red) / Untreated control (Green) cells labeling gamma H2A.X (phospho S139) with ab303656 at 1/1000 dilution (0.1µg) compared with a mouse monoclonal IgG (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue).Goat anti-Mouse IgG (Alexa Fluor® 488 ab150113) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labeling gamma H2A.X (phospho S139) with ab303656 at 1/500 dilution followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green).Confocal image showing increased nuclear staining in HeLa cells treated with UV-C (50 mJ/cm2) then recovery (1h).ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% PFA-fixed 0.1% Triton X-100 permeabilized UV-treated HCT116 cells labelling Rad51 with ab133534 at 0.2 μg/ml (shown in green). ab303656 Anti-gamma H2A.X (phospho S139) antibody [N1-431] was used as a DNA-damage counterstain at 0.2 μg/ml (shown in red). The secondary antibodies used were ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed and ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed, both were used at 1/1000 (2 μg/ml) dilution. Image shows selected areas of overlapping foci. The nuclear counterstain was DAPI.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling gamma H2A.X (phospho S139) with ab303656 at 1/500 dilution followed by ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green).Confocal image showing increased nuclear staining in NIH/3T3 cells treated with UV-C (50 mJ/cm2) then recovery (1h).ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/50 dilution (10 µg/mL) (Red). The nuclear counterstain was DAPI (Blue).Secondary antibody only control : Secondary antibody is ab150117 Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

• WB

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Western blot - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID : 11673449 and 11571274 ).

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-gamma H2A.X (phospho S139) antibody [N1-431] (<a href='/en-us/products/primary-antibodies/gamma-h2ax-phospho-s139-antibody-n1-431-ab303656'>ab303656</a>) at 1/1000 dilution

Lane 1:

Untreated HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

HeLa treated with 50mJ/cm2 UV-C, then recovery for 30 minutes, whole cell lysate at 20 µg

Lane 3:

Untreated Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 20 µg

Lane 4:

Jurkat treated with 25uM etoposide for 8 hours, whole cell lysate at 20 µg

Lane 5:

Untreated NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 20 µg

Lane 6:

NIH/3T3 treated with 100mJ/cm2 UV-C, then recovery for 2 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/2000 dilution

Observed band size: 15 kDa

false

Exposure time: 3min

• PepArr

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Peptide Array - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Peptide array analysis of ab303656 at 0.05 µg/ml followed by a Goat Anti-Rabbit IgG (H+L) Fluor 647nm conjugated at 1/50000 dilution. All batches of ab303656 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles all other peptides as blue circles. The affinity is calculated as the area under the curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Product Protocol section.

• Dot

Supplier Data

Dot Blot - Anti-gamma H2A.X (phospho S139) antibody [N1-431] - BSA and Azide free (AB325973)

This data was developed using ab303656, the same antibody clone in a different buffer formulation.

Dot blot analysis of gamma H2A.X (phospho S139) using ab303656 at 1/1000 dilution followed by a Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution. Lane 1 : H2A.X (phospho S139) peptide. Lane 2 : H2A.X non-phospho peptide. Exposure time : 3 minutes. Blocking and diluting buffer and concentration : 5% NFDM/TBST.