Anti-Histone H2A.X/H2AFX Antibody Picoband®

Western blot analysis of H2AFX using anti-H2AFX antibody (A00241-1).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human HEL whole cell lysates,
Lane 2: human PC-3 whole cell lysates,
Lane 3: human CACO-2 whole cell lysates,
Lane 4: rat PC-12 whole cell lysates,
Lane 5: rat C6 whole cell lysates,
Lane 6: mouse NIH/3T3 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-H2AFX antigen affinity purified polyclonal antibody (Catalog # A00241-1) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for H2AFX at approximately 15 kDa. The expected band size for H2AFX is at 15 kDa.

IHC analysis of H2AFX using anti-H2AFX antibody (A00241-1).
H2AFX was detected in a paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-H2AFX Antibody (A00241-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

IHC analysis of H2AFX using anti-H2AFX antibody (A00241-1).
H2AFX was detected in a paraffin-embedded section of mouse liver tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-H2AFX Antibody (A00241-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Immunoprecipitating (IP) H2AFX in 293T whole cell lysate.
Western blot analysis of H2AFX using anti-H2AFX antibody (A00241-1);
Lane 1: 293T whole cell lysates (30ug);
Lane 2: Rabbit control IgG instead of anti-H2AFX antibody in 293T whole cell lysate;
Lane 3: anti-H2AFX antibody (2μg) + 293T whole cell lysate (500μg).
After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-H2AFX antigen affinity purified polyclonal antibody (A00241-1) at a dilution of 0.5 μg/mL and probed with a goat anti-rabbit IgG-HRP secondary antibody (Catalog # BA1054). The signal is developed using ECL Plus Western Blotting Substrate (Catalog # AR1196-200). A specific band was detected for H2AFX at approximately 15 kDa. The expected band size for H2AFX is at 15 kDa.

Flow Cytometry analysis of PC-3 cells using anti-H2AFX antibody (A00241-1).
Overlay histogram showing PC-3 cells stained with A00241-1 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-H2AFX Antibody (A00241-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.