GALC Antibody / Galactocerebrosidase (FY13064)

Western blot analysis of GALC using anti-GALC antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human whole cell lysates, Lane 2: human whole cell lysates, Lane 3: human SH-SY5Y whole cell lysates, Lane 4: human HEL whole cell lysates, Lane 5: rat brain tissue lysates, Lane 6: mouse brain tissue 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-GALC antibody at 1:1000 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. A single major band is detected at approximately 100 kDa, higher than the predicted molecular weight of 77 kDa. This upward shift is well documented for GALC and reflects its heavily N-glycosylated precursor form. The enzyme is synthesized as a ~100 kDa glycoprotein that is subsequently cleaved within lysosomes into smaller subunits, although only the full-length precursor is prominent in whole-cell lysates.