Figure 1. Western blot analysis of Nkx2.5/NKX2-5 using anti-Nkx2.5/NKX2-5 antibody (A00738-2). 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 30 ug of sample under reducing conditions. Lane 1: human U20S whole cell lysates, Lane 2: human U251 whole cell lysates, Lane 3: human SiHa whole cell lysates. 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-Nkx2.5/NKX2-5 antigen affinity purified polyclonal antibody (Catalog # A00738-2) 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 1:5000 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 Nkx2.5/NKX2-5 at approximately 35 kDa. The expected band size for Nkx2.5/NKX2-5 is at 35 kDa.