Western blot analysis of Atg4A using anti-Atg4A antibody (M06539).
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 HepG2 whole cell lysates,
Lane 2: human Hela whole cell lysates,
Lane 3: human Jurkat whole cell lysates,
Lane 4: human T-47D whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: mouse brain tissue lysates,
Lane 7: mouse C2C12 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-Atg4A antigen affinity purified monoclonal antibody (Catalog # M06539) at 1:500 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 Atg4A at approximately 45 kDa. The expected band size for Atg4A is at 45 kDa.