In-vitro GCase Activity Assay

Mix 20mL (0.852g in 30mL) with 14mL (0.576g in 30mL) to make M-buffer (5.4, no additional pH adjustment required).

Dissolve one protease inhibitor tablet (Roche cOmplet mini) in 10mL, then add Triton X-100 solution to 0.25% (v/v) (e.g. 25µL in 10mL) and0.2Mass / % volume of sodium taurocholate to make active GCase buffer.

Set up the desired plate layout in a 384 well plate (flat bottom, black). There should be two sections, as each sample must be prepared and assayed both with and without CBE (CBE: GCase1 inhibitor).

Prepare 0.8millimolar (mM) by diluting 10millimolar (mM) in DMSO with the GCase buffer (GCase buffer:CBE = 92:8).

Prepare CBE-free carrier solution in the same volume of GCase buffer/DMSO (GCase buffer:DMSO = 92:8).

Pipette 10µL diluted with GCase buffer into wells of a 384- well plate. Four replication sets should be run (sample concentration: 0.7mg/mL ~ 1.2mg/mL). Protein concentration should be adjusted to be similar between control and experiment groups using GCase buffer.

Add 5µL to the CBE-positive wells or the same volume of CBE-free carrier solution to the CBE-negative wells.

Cover the plate with aluminum foil and briefly centrifuge. Incubate shaking: 600rpm.

During incubation, prepare 4-Methylumbelliferyl-B-D-glucoside (4-MU) diluent by diluting 1 M 4-Mu (338 mg per 1ml DMF) with GCase buffer to a final concentration of 2.5millimolar (mM) (1:400 dilution).

After the CBE incubation, spin down the plate and add 15µL to reach a total volume of 30µL in each well.

Cover the plate with aluminum and briefly centrifuge, and Incubate shaking: 450rpm.

Prepare stop solution by adding 4mL and 1.877g up to 25mL in water. Final concentration of glycine is 1Molarity (M) (pH 10.5)

After incubation, spin down the plate again and Add 30µL to each well.

Read the 4-MU fluorescence with a microplate reader (Excitation: 365 nm; Emission: 449 nm; Cutoff: 435nm; 3 reads/well).

GCase activity in each protein lysate can be calculated as below.