Odyssey image scanner

Rat brain area homogenates (100μg) were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA). The blots were blocked in 2% (w/v) non-fat dried milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% (v/v) Tween-20) and incubated with α-GBA (Sigma, St. Louis, USA), α-GBA2 (Eurogentec custom made) and α-Tubulin (Cedarlane Laboratories, Ontario, Canada) primary antibodies diluted in blocking buffer, overnight at 4°C. Blots were washed for 30 minutes in TBST. After washing, the membranes were incubated with matching IRDye-conjugated secondary antibodies (Westburg BV, Leusden, Netherlands) for 1 hour at room temperature. Blots were scanned on an Odyssey image scanner (GE Healthcare).

Equal amounts of protein (50 μg) were subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The blots were blocked in 5% (w/v) nonfat dried milk in TBST buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% [v/v] Tween-20) and probed with anti-GBA2 (1:1,000), anti-GBA (1:1,000) or anti-tubulin (clone DM1A, ascites fluid, 1:10,000, Sigma-Aldrich, St Louis, MO, USA) antibody diluted in blocking buffer, overnight at 4°C. After washing, the membranes were incubated with secondary antibody (anti-rabbit/mouse IgG IRDye 800CW [Westburg, Leusden, The Netherlands]) diluted 1:10,000 in blocking buffer, for 1 h at RT. Blots were scanned on an Odyssey image scanner (GE Healthcare, Munich, Germany).