Western blot analysis of AMPK alpha 1 using anti-AMPK alpha 1 antibody (A00994-3).
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 SiHa whole cell lysates,
Lane 2: human Hela whole cell lysates,
Lane 3: human RT4 whole cell lysates,
Lane 4: human A549 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat PC-12 whole cell lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse NIH/3T3 whole cell 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-AMPK alpha 1 antigen affinity purified polyclonal antibody (Catalog # A00994-3) 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 1:5000 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 AMPK alpha 1 at approximately 64 kDa. The expected band size for AMPK alpha 1 is at 64 kDa.