Nov 07, 2025

GCase activity assay

DOI
https://dx.doi.org/10.17504/protocols.io.3byl46yb8go5/v1
  • Sara Lucas Del Pozo1,2,
  • Giuseppe Uras1,2,3,
  • Federico Fierli1,2,
  • Veronica Lentini1,3,
  • Sofia Koletsi1,2,
  • Carlos Lazaro-Hernandez1,4,
  • Kai-Yin Chau1,2,
  • Derralynn A. Hughes5,
  • Anthony H.V. Schapira5
  • 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
  • 3Department of Biomedical Science, University of Sassari, Viale San Pietro, Sassari 07100, Italy;
  • 4Neurology Department, Vall d'Hebron University Hospital, Barcelona, Spain;
  • 5Lysosomal Storage Disorders Unit, Royal Free Hospital NHS Foundation Trust and University College London, London, UK
Sofia Koletsi
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl46yb8go5/v1
Protocol Citation: Sara Lucas Del Pozo, Giuseppe Uras, Federico Fierli, Veronica Lentini, Sofia Koletsi, Carlos Lazaro-Hernandez, Kai-Yin Chau, Derralynn A. Hughes, Anthony H.V. Schapira 2025. GCase activity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46yb8go5/v1
Manuscript citation:
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2023
Last Modified: November 07, 2025
Protocol  Integer ID: 231665
Keywords: ASAPCRN, gcase protein, gcase activity, crucial part of the assay, acidic enzyme assay buffer, glucopyranoside, enzyme, lysosome, assay, enzyme in an active confirmation, utilised bile salt, bile salt, low ph, blue fluorogenic substrate, utilising blue fluorogenic substrate, taurocholate
Funders Acknowledgements:
Aligning Science Across Parkinson's Disease
Grant ID: ASAP-000420
Abstract
This protocol describes a commonly used approach to measure GCase activity by utilising blue fluorogenic substrate 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG). In order to simulate the lysosome's low pH, this method uses GCase protein that has been isolated from a cellular model and diluted in an acidic enzyme assay buffer. The inclusion of a lipid or detergent to keep the enzyme in an active confirmation is a crucial part of the assay. Taurocholate, a regularly utilised bile salt, is used in this protocol.
Materials
Assay Buffer:
AB
Citric acid50 mM
K2HPO4176 mM
Sodium Taurocholate (Sigma Aldrich REF 86339-5G)10 mM
Tween 20 (Sigma Aldrich REF P9416-50ML)0.01%
dH2O
NOTE: The pH should adjusted down to make it closer to pH 5.8
Complete Lysis Buffer:
AB
Cold Assay Buffer10 ml
20% Triton X (Sigma Aldrich‱ PCode: 1002733102)100 µl
Protease Cocktail Inhibitor (Thermob Scientific REF A32965)100 µl
STOP solution:
NaOH0.5 M
Glycine0.5 M
NOTE: The solution need to be adjusted to 1000ml with distilled H2O (ie addition of 650ml) and the solution also needs to be adjusted to a pH of 10 approximately. The solution could be stored at room temperature.
Substrate:

1 millimolar (mM) 4-methylumbelliferyl β-D glucopyranosidase (4-MUG) (Sigma Aldrich‱ REF M3633-250MG)
Note
Dilute 4MUG in Assay Buffer.
Sodium taurocholate hydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #86339
TWEEN® 20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P9416
Pierce™ Protease Inhibitor Tablets, EDTA-freeThermo FisherCatalog #A32965
4-Methylumbelliferyl β-D-glucopyranosideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3633
Phosphate buffered saline (PBS) without Ca/Mg Thermo Fisher ScientificCatalog #14190144

GCase activity assay
1d 1h 20m
Seed the total of 3*104 cells in each well of a 96-well plate (Corning® Assay Plate REF 3340) and left to incubate for 24:00:00 .

1d
Remove the cell medium and wash each well 2 times with 1X PBS (Gibco® REF 14190-144) at Room temperature

Lyse the cells using 50 µL cold Complete Lysis Buffer for 00:10:00 at 4 °C in a shaker.

10m
Afterwards, wash the cells two times in 200 µL of sterile 1X PBS.

Subsequently, supplement 50 µL of Assay Buffer with 1 millimolar (mM) 4-MUG that are added to each well and left the samples to incubate Overnight at Room temperature and protected from light.

10m
Stop the reaction by adding 100 µL of STOP solution to each well, and then is left to incubate for 01:00:00 at Room temperature .

1h
Analyse the plates using CytationTM® plate reader using endpoint protocol with wavelength ex/em set up at 360/460nm.
Normalise the obtained data to control and expresses as a fraction.