Western blot analysis of GAPDH using anti-GAPDH antibody (A00227-HRP).
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 A549 whole cell lysates,
Lane 3: human A562 whole cell lysates,
Lane 4: human MCF-7 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat liver tissue lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse liver 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-GAPDH antigen affinity purified polyclonal antibody (Catalog # A00227-HRP) at 0.1 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for GAPDH at approximately 36 kDa. The expected band size for GAPDH is at 36 kDa.