Western blot analysis of PKC alpha using anti-PKC alpha antibody (PB9318).
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 U87 whole cell lysates,
Lane 2: human HEL whole cell lysates,
Lane 3: human A549 whole cell lysates,
Lane 4: human U20S whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: mouse brain 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-PKC alpha antigen affinity purified polyclonal antibody (Catalog # PB9318) 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 PKC alpha at approximately 77 kDa. The expected band size for PKC alpha is at 80 kDa.