Figure 1. Western blot analysis of API5 using anti-API5 antibody (PA1009). 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 30 ug of sample under reducing conditions. Lane 1: Rat Cardiac Muscle Tissue Lysate Lane 2: Rat Brain Tissue Lysate, Lane 3: Rat Testis Tissue Lysate, Lane 4: Rat Placenta Tissue Lysate, Lane 5: MCF-7 Cell Lysate, Lane 6: HELA Cell Lysate, Lane 7: CEM Cell Lysate, Lane 8: SMMC Cell Lysate, Lane 9: COLO320 Cell Lysate. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA 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-API5 antigen affinity purified polyclonal antibody (Catalog # PA1009) 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 1:5000 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 API5 at approximately 59 kDa. The expected band size for API5 is at 59 kDa.