Figure 1. Western blot analysis of Nedd4 using anti-Nedd4 antibody (A00984-4). 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: rat L6 whole cell lysates, Lane 2: mouse skeletal muscle tissue lysates, Lane 3: mouse lung tissue lysates, Lane 4: mouse B16-F10 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-Nedd4 antigen affinity purified polyclonal antibody (Catalog # A00984-4) at 0.25 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 Nedd4 at approximately 115 kDa. The expected band size for Nedd4 is at 149 kDa.