Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)
Key features and details
- Rabbit polyclonal to Histone H2A.X - Nuclear Marker
- Suitable for: WB, IHC-P
- Knockout validated
- Reacts with: Cow, Human
- Isotype: IgG
Overview
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Product name
Anti-Histone H2A.X antibody - Nuclear Marker
See all Histone H2A.X primary antibodies -
Description
Rabbit polyclonal to Histone H2A.X - Nuclear Marker -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species IHC-P HumanWB CowHuman
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Immunogen
Synthetic peptide corresponding to Human Histone H2A.X aa 100 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin. Synthetic peptide derived from residues 100 - 200 of Human H2A.X.
Read Abcam's proprietary immunogen policy .
Database link: P16104
(Peptide available asab15020) -
General notes
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.
One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.
Learn more here.
Images
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Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)All lanes : Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution
Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : H2AFX (Histone H2A.X) knockout HAP1 whole cell lysate
Lane 3 : HEK293 whole cell lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 15 kDaLanes 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. -
Western blot - Anti-Histone H2A.X antibody - Nuclear Marker (ab10475)Lanes 1 & 3 : Anti-Histone H2A.X antibody - Nuclear Marker (ab10475) at 1/500 dilution
Lane 2 : 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) at 1 µg
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size: 15 kDa
Exposure time: 30 seconds
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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 BondTM 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.
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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.
