Anti-Histone H2A.X 抗体 - Nuclear Marker

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2A.X antibody - Nuclear Marker (AB10475)

IHC image of Histone H2A X staining in human B cell lymphoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab10475, 1μg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

• WB

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Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (AB10475)

HeLa 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.

All lanes:

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)

Predicted band size: 15 kDa

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• WB

Lab

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (AB10475)

Lanes 1 - 4 : Merged signal (red and green). Green - ab10475 observed at 17 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab10475 was shown to specifically react with Histone H2A.X in wild-type HAP1 cells as signal was lost in H2AFX (Histone H2A.X) knockout cells. Wild-type and H2AFX (Histone H2A.X) knockout samples were subjected to SDS-PAGE. ab10475 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

H2AFX (Histone H2A.X) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HEK293 whole cell lysate at 20 µg

Predicted band size: 15 kDa

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• WB

Supplier Data

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (AB10475)

Lanes 1 and 3:

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution

Lane 2:

Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/1000 dilution

Lanes 1 - 2:

Calf thymus histone lysate

Lane 3:

Calf thymus histone lysate with Human Histone H2A.X (unmodified ) peptide (ab15020)

Secondary

All lanes:

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

Predicted band size: 15 kDa

Observed band size: 15 kDa

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Exposure time: 30s