Western blot analysis of Phospho-Smad2 (S250) using anti-Phospho-Smad2 (S250) antibody (P00090-1).
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 Hela whole cell lysates,
Lane 2: human HepG2 whole cell lysates,
Lane 3: human HT-1080 whole cell lysates,
Lane 4: rat heart tissue lysates,
Lane 5: rat skeletal muscle tissue lysates,
Lane 6: mouse heart tissue lysates,
Lane 7: mouse skeletal muscle tissue 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-Phospho-Smad2 (S250) antigen affinity purified monoclonal antibody (Catalog # P00090-1) at 1:1000 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 1:1000 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 Phospho-Smad2 (S250) at approximately 58 kDa. The expected band size for Phospho-Smad2 (S250) is at 52 kDa.