Western blot analysis of Raf1 in, Lane 1: rat kidney tissue lysate, Lane 2: mouse kidney tissue lysate, Lane 3: human MCF-7 whole cell lysate, Lane 4: human 22RV1 whole cell lysate, Lane 5: human Hela whole cell lysate. 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 50 µg of sample under reducing conditions. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. The membrane was blocked with 5% Non-fat Milk/ TBS for 1. 5 hour at RT. The membrane was incubated with c-Raf Polyclonal Antibody (Product # PA5-95464) at 0.5 μg/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. A specific band was detected for Raf1 at approximately 73KD. The expected band size for Raf1 is at 73KD.