Western blot analysis of BAD expression in (1) HeLa cell lysate; (2) HeLa cell treated with Calcyculin A lysate using anti-BAD antibody (MP03520). 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-BAD antigen affinity purified polyclonal antibody (Catalog # MP03520) 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 # SA1022) with Tanon 5200 system. A specific band was detected for BAD.