Western blot analysis of TFEB using anti-TFEB antibody (A00662-2).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human K562 whole cell lysates,
Lane 2: human 293T whole cell lysates,
Lane 3: human Hela whole cell lysates,
Lane 4: human Jurkat whole cell lysates,
Lane 5: human HepG2 whole cell lysates,
Lane 6: human Raji whole cell lysates,
Lane 7: human A549 whole cell lysates,
Lane 8: human SW620 whole cell lysates.
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-TFEB antigen affinity purified polyclonal antibody (Catalog # A00662-2) 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 ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for TFEB at approximately 65 kDa. The expected band size for TFEB is at 53 kDa.