Figure 1. Western blot analysis of Semaphorin 3B/SEMA3B using anti-Semaphorin 3B/SEMA3B antibody (M06559). 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 Hela whole cell lysates, Lane 2: human CACO-2 whole cell lysates, Lane 3: human U87 whole cell lysates, Lane 4: human K562 whole cell lysates, Lane 5: rat kidney tissue lysates, Lane 6: rat NRK whole cell lysates, Lane 7: mouse kidney tissue lysates, Lane 8: mouse ANA-1 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 mouse anti-Semaphorin 3B/SEMA3B antigen affinity purified monoclonal antibody (Catalog # M06559) 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-mouse 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 # EK1001) with Tanon 5200 system. A specific band was detected for Semaphorin 3B/SEMA3B at approximately 83 kDa. The expected band size for Semaphorin 3B/SEMA3B is at 83 kDa.