Figure 1. Western blot analysis of CDK4 using anti-CDK4 antibody (PA1428-1, Left) and anti-CDK4 antibody (PB9535, Right). 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 K562 whole cell lysates, Lane 3: rat PC-12 whole cell lysates, Lane 4: mosue NIH/3T3 whole cell lysates, Lane 5: mosue RAW264.7 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-CDK4 antigen affinity purified polyclonal antibody (Catalog # PA1428-1) and rabbit anti-CDK4 antigen affinity purified polyclonal antibody (Catalog # PB9535)at 0.5 microg/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 CDK4 at approximately 34 kDa. The expected band size for CDK4 is at 34 kDa.