Western blot analysis of CDK4 using anti-CDK4 antibody (M00159).
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 Hela whole cell lysates,
Lane 2: human HepG2 whole cell lysates,
Lane 3: human MCF-7 whole cell lysates,
Lane 4: human K562 whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: rat pancreas tissue lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse pancreas 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-CDK4 antigen affinity purified monoclonal antibody (Catalog # M00159) at 1:1000 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 1:1000 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 CDK4 at approximately 34 kDa. The expected band size for CDK4 is at 34 kDa.