Glucocerebrosidase Activity Assay

Cells were resuspended in homogenization buffer (250 mM Sucrose, 10 mM Tris pH 7.5, 1 mM EDTA, 025% Triton X-100) and the cell pellet was disrupted on ice with a probe sonicator thrice for 5 s at 50 W. The cell lysate was centrifuged at 20,000 g for 20 min at 4°C, protein concentration quantified and normalized to 1 μg/μl. In a 96-well plate, 25 μl/well cell lysate, 100 μl of assay buffer (0.2 M sodium phosphate dibasic, 0.1 M citric acid), 25 μl of 4-methylumbelliferyl β-D-glucopyranoside substrate, incubated for 30 min at 37°C, reaction stopped with 75 μl stop solution (1 M Glycine, pH 10.5) and fluorescence read at 355 nm excitation 450 emission.