TPBG Antibody / Trophoblast glycoprotein (FY12461)

Western blot analysis of TPBG using anti-TPBG antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human MCF-7 whole cell lysates, Lane 2: human Hacat whole cell lysates, Lane 3: human RT4 whole cell lysates, Lane 4: human PC-3 whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: rat PC-12 whole cell lysates, Lane 7: mouse brain tissue lysates, Lane 8: mouse NIH/3T3 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-TPBG antibody at 0.5 ug/ml overnight at 4oC, 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 was developed using an ECL Plus Western Blotting Substrate. TPBG (~45 kDa predicted) was detected primarily at ~70-80 kDa, consistent with extensive N-linked glycosylation of the extracellular domain. A faint higher band (~90-100 kDa) likely represents a hyper-glycosylated variant of TPBG, as previously reported for the trophoblast glycoprotein 5T4.