Figure 1. Western blot analysis of NEDD4-2 using anti-NEDD4-2 antibody (A01595-1). 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 50ug of sample under reducing conditions. Lane 1: human 22RV1 whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: rat pancreas tissue lysates, Lane 4: mouse pancreas tissue lysates, Lane 5: mouse NIH3T3 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA 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-NEDD4-2 antigen affinity purified polyclonal antibody (Catalog # A01595-1) 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 1:10000 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 NEDD4-2 at approximately 130KD. The expected band size for NEDD4-2 is at 112KD.