Western blot analysis of CD47 using anti-CD47 antibody (A00360-1). 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 MCF-7 whole cell lysates, Lane 2: human SK-OV-3 whole cell lysates, Lane 3: human PANC-1 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-CD47 antigen affinity purified polyclonal antibody (Catalog # A00360-1) at 0.5 ug/mL overnight at 4 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 CD47 at approximately 41KD. The expected band size for CD47 is at 35KD.