Western blot analysis of Raptor/RPTOR using anti-Raptor/RPTOR antibody (A01463-3).
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 30ug of sample under reducing conditions.
Lane 1: human HepG2 whole cell lysates,
Lane 2: human Hela whole cell lysates,
Lane 3: human Mcf-7 whole cell lysates,
Lane 4: human Hek293 whole cell lysates,
Lane 5: human A549 whole cell lysates,
Lane 6: rat brain tissue lysates,
Lane 7: mouse brain tissue 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-Raptor/RPTOR antigen affinity purified polyclonal antibody (Catalog # A01463-3) 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 Raptor/RPTOR at approximately 150KD. The expected band size for Raptor/RPTOR is at 150KD.