Phospho-Histone H3 (T3) H3F3A Monoclonal Antibody

IHC analysis of H3F3A using anti-H3F3A antibody (orb548536). H3F3A 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-H3F3A Antibody (orb548536) overnight at 4°C. Biotinylated goat anti Rabbit IgG IgG antibody 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 H3F3A expression in HeLa cell lysates treated with FBS + Calyculin A using anti-H3F3A antibody (orb548536). 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-H3F3A antigen affinity purified polyclonal antibody (Catalog # orb548536) 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 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 # orb90444) with Tanon 5200 system. A specific band was detected for H3F3A.