Western blot analysis of Cytochrome C using anti-Cytochrome C antibody. 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 lysate,Lane 2: rat heart tissue lysate,Lane 3: mouse brain tissue lysate,Lane 4: mouse heart tissue lysate,Lane 5: human Hela whole Cell lysate,Lane 6: human MCF-7 whole Cell lysate,Lane 7: human 293T whole Cell lysate,Lane 8: human HepG2 whole Cell lysate,Lane 9: human Jurkat whole Cell lysate. 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-Cytochrome C antigen affinity purified polyclonal antibody 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 with Tanon 5200 system. A specific band was detected for Cytochrome C at approximately 14KD. The expected band size for Cytochrome C is at 12KD.