Phospho-Histone H2A.X (S139) H2AFX Monoclonal Antibody

• Catalog Number: orb548529
• Category: Antibodies
• Description: Phospho-Histone H2A.X (S139) H2AFX Monoclonal Antibody
• Species/Host: Rabbit
• Clonality: Monoclonal
• Clone Number: AbH41
• Tested applications: ICC, IF, IHC, IP, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: A synthesized peptide derived from human Phospho-Histone H2A.X (S139)
• Concentration: Actual concentration vary by lot. Use suggested dilution ratio to decide dilution procedure.
• Dilution range: WB 1:5000-1:10000IHC 1:50-1:200ICC/IF 1:50-1:200IP 1:30
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: 15 kDa
• UniProt ID: P16104
• Storage: Store at -20°C for one year. For short term storage and frequent use, store at 4°C for up to one month. Avoid repeated freeze-thaw cycles.
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Immunofluorescent analysis of HeLa cells treated with H2O2, using Phospho-Histone H2A.X (S139) Antibody.

IHC analysis of H2AFX using anti-H2AFX antibody (orb548529). H2AFX was detected in paraffin-embedded section. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-H2AFX Antibody (orb548529) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

Western blot analysis of H2AFX expression in Jurkat cell lysate treated with etoposide using anti-H2AFX antibody (orb548529). 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 50ug of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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 # orb548529) at 0.5 ug/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 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # orb90503) with Tanon 5200 system.