Figure 1. Western blot analysis of NOS3 using anti-NOS3 antibody (A01604-2). 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 placenta tissue lysates Lane 2: human K562 whole cell lysates Lane 3: human HepG2 whole cell lysates Lane 4: human THP-1 whole cell lysates Lane 5: rat kidney tissue lysates Lane 6: mouse kidne tissue lysates Lane 7: mouse small intestine 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 anti-NOS3 antigen affinity purified polyclonal antibody (Catalog # A01604-2) 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 NOS3 at approximately 133KD. The expected band size for NOS3 is at 133KD.