Mar 17, 2023
In vitro GCase activity assay (total cell lysate)
- Federico Bertoli1,
- Michela Deleidi1
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: Federico Bertoli, Michela Deleidi 2023. In vitro GCase activity assay (total cell lysate). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvordxbv4o/v1
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: March 08, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 78340
Keywords: In vitro GCase activity assay, total cell lysate, ASAPCRN
Abstract
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor).
Attachments
ggmvbqjbx.pdf
615KB
Materials
Reagents
- 4-Methylumbelliferyl β-D-glucopyranosideSigma – AldrichCatalog #M3633
- Conduritol-b-epoxideMerck MilliporeCatalog #234599
- AMP-Deoxynojirimycin (CAS 216758-20-2)Catalog #sc-223780
- 1%Triton Base Buffer:
| A | B | C | |
| 1% Triton Base Buffer | Final concentration | Amount | |
| Triton X-100 | 1% | 0.5 mL | |
| 5 M NaCl | 150 mM | 1.5 mL | |
| 1 M HEPES pH 7.4 | 20 mM | 1 mL | |
| 0.5 M EDTA | 1 mM | 100 μL | |
| 1 M MgCl2 | 1.5 mM | 75 μL | |
| 100% glycerol | 10% | 5 mL | |
| Milli-Q H2O | n/a | 41.825 mL |
- 1% Triton extraction buffer:
| A | B | C | |
| 1% Triton Extraction Buffer | Final concentration | Amount | |
| 1% Triton Base Buffer | n/a | 4.425 mL | |
| PIC | n/a | ½ tablet | |
| 500 mM NaF | 50 mM | 500 μL | |
| 200 mM Na3VO4 | 2 mM | 50 μL | |
| 0.1 M PMSF | 0.5 mM | 25 μL |
- McIlvaine Buffer:
| A | B | C | |
| pH | 0.2 M NaHPO4 (mL) | 0.1 M citric acid (mL) | |
| 6.0 | 12.63 | 7.37 |
Sample Lysis
Sample Lysis
Suspend samples in 50 µL of 1% Triton extraction buffer.
Homogenize with a Dounce homogenizer for 25 strokes.
Rotate samples for 00:30:00 at 4 °C .
30m
Centrifuge at 13500 x g , 4 °C for 00:15:00 .
15m
Collect supernatants.
Substrate preparation
Substrate preparation
Add 20.30 mg 4-Methylumbelliferyl-β-D-glucopyranoside for 10 mL ddH2O of substrate (6 millimolar (mM) ).
Incubate at 55 °C and vortex every 00:05:00 until dissolved (approx. 00:30:00 ).
35m
Store at 4 °C until needed.
Sample preparation
Sample preparation
Add the equivalent of 10 µg total protein in ddH2O to reach a final 45 µL volume.
Note
For each sample
Add to each 25 µL McIlave Buffer 6 and mix it.
Note
For GBA2 inhibition, 5 nanomolar (nM) AMP-Deoxynojirimycin
Divide the overall 70 µL volume into two tubes (35 µL each).
Incubate one tube with 5 µL CBE 1 millimolar (mM) at Room temperature for 00:30:00 .
30m
Incubate the other one with 5 µL ddH2O at Room temperature for 00:30:00 .
30m
Enzymatic reaction
Enzymatic reaction
Add 25 µL substrate to each reaction tube.
Incubate at 37 °C for 02:00:00 .
2h
Measurement
Measurement
Take 10 µL of each reaction tube into a 96-well plate (in triplicate).
Add 90 µL 0.2 Molarity (M) glycine 10.2 to each well to stop the reaction.
Measure fluorescence: Excitation 355nm, Emission 460nm.
Note
GBA1 activity is obtained by subtracting the background and GBA2 activity from the total GCase activity.