Western blot analysis of NEDD8 using anti-NEDD8 antibody (M00547).
Electrophoresis was performed on a 12% 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 MCF-7 whole cell lysates,
Lane 3: human RT4 whole cell lysates,
Lane 4: human PC-3 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat heart tissue lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse heart 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-NEDD8 antigen affinity purified monoclonal antibody (M00547) 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 1:500 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for NEDD8 at approximately 10 kDa. The expected band size for NEDD8 is at 9 kDa.