Western blot analysis of SMAD1 using anti-SMAD1 antibody (A00728-1). 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 50ug of sample under reducing conditions. Lane 1: rat kidney tissue lysate, Lane 2: rat testis tissue lysate, Lane 3: mouse brain tissue lysate, Lane 4: mouse kidney tissue lysate, Lane 5: mouse testis tissue lysate, Lane 6: human HeLa cell lysate, Lane 7: human A375 cell lysate, Lane 8: human HepG2 cell lysate. 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-SMAD1 antigen affinity purified polyclonal antibody (Catalog # A00728-1) 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 1:10000 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 SMAD1 at approximately 52,60KD. The expected band size for SMAD1 is at 48, 52, 60KD.