Figure 2. Western blot analysis of EPS15 using EPS15. 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: rat brain tissue lysates, Lane 2: rat liver tissue lysates, Lane 3: mouse brain tissue lysates, Lane 4: mouse spleen tissue lysates, Lane 5: mouse liver tissue lysates, Lane 6: mouse testis tissue 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 EPS15 at 0.5ug/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat 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 with Tanon 5200 system. A specific band was detected for EPS15 at approximately 140kD. The expected band size for EPS15 is at 99kD.