Figure 1. Western blot analysis of ALYREF using anti-ALYREF antibody (A03580-3). 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 placenta tissue lysates, Lane 2: human PC-3 whole cell lysates, Lane 3: human Hela whole cell lysates, Lane 4: human HepG2 whole cell lysates, Lane 5: human 293T whole cell lysates, Lane 6: human A431 whole cell lysates, Lane 7: human A549 whole cell lysates, Lane 8: rat brain tissue lysates, Lane 9: rat liver tissue lysates, Lane 10: rat PC-12 whole cell lysates, Lane 11: rat NRK whole cell lysates, Lane 12: mouse brain tissue lysates, Lane 13: mouse liver tissue lysates, Lane 14: mouse Neuro-2a whole cell lysates, Lane 15: mouse RAW264.7 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-ALYREF antigen affinity purified polyclonal antibody (Catalog # A03580-3) 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 ALYREF at approximately 35 kDa. The expected band size for ALYREF is at 27 kDa.