Western blot analysis of Parkin using anti-Parkin antibody (PB9307).
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 293T whole cell lysates,
Lane 2: human U251 whole cell lysates,
Lane 3: rat brain tissue lysates,
Lane 4: rat C6 whole cell lysates,
Lane 5: rat PC-12 whole cell lysates,
Lane 6: mouse brain tissue lysates,
Lane 7: mouse Neuro-2a 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-Parkin antigen affinity purified polyclonal antibody (PB9307) 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 (Catalog # BA1054) 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 Parkin at approximately 52 kDa. The expected band size for Parkin is at 52 kDa.