Aug 01, 2023
In-vitro GCase Activity Assay
- 1Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, USA.
Protocol Citation: Tae-Un Han, sidranse 2023. In-vitro GCase Activity Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3b7dvzp/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: August 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85785
Keywords: ASAPCRN, GCase, GCase activity
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000458
Abstract
This is version 2 of in-vitro GCase activity assay.
We optimized the assay condition to enhance sensitivity and specificity.
Both cell lysates and protein lysates prepared in M-buffer containing protease inhibitor and 0.25% TritonX can use this protocol.
Attachments
Materials
- Cell lysates and protein lysates prepared in M-buffer containing protease inhibitor and 0.25% TritonX
- 0.2M Na2HPO4
- 0.1M citrate
- protease inhibitor tablet (Roche cOmplet mini)
- Triton X-100 solution
- 384 well plate (flat bottom, black)
- 10 mM CBE (5 mg CBE in 3086 ul DMSO)
- DMSO
- aluminum foil
- sodium taurocholate powder
- 1 M 4-Methylumbelliferyl-B-D-glucoside (4-MU-G, 338 mg per 1ml DMF)
- 5 M NaOH
- glycine
- microplate reader
Mix 20 mL 0.2M Na2HPO4 (0.852 g in 30 mL water ) with 14 mL 0.1M citrate (0.576 g in 30 mL water ) to make M-buffer (5.4 , no additional pH adjustment required).
Dissolve one protease inhibitor tablet (Roche cOmplet mini) in 10 mL M-buffer , then add Triton X-100 solution to 0.25 % (v/v) (e.g. 25 µL in 10 mL ) and0.2 Mass / % 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 0.8 millimolar (mM) CBE by diluting 10 millimolar (mM) CBE 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 10 µL protein lysate diluted with GCase buffer into wells of a 384- well plate. Four replication sets should be run (sample concentration: 0.7 mg/mL ~ 1.2 mg/mL ). Protein concentration should be adjusted to be similar between control and experiment groups using GCase buffer.
Add 5 µL 0.8 mM CBE solution 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: 600 rpm, 37°C, 00:15:00 .
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 2.5 millimolar (mM) (1:400 dilution).
After the CBE incubation, spin down the plate and add 15 µL assay buffer with 2.5 mM 4-MU to reach a total volume of 30 µL in each well.
Cover the plate with aluminum and briefly centrifuge, and Incubate shaking: 450 rpm, 37°C, 01:00:00 .
Prepare stop solution by adding 4 mL 5M NaOH and 1.877 g glycine up to 25 mL in water. Final concentration of glycine is 1 Molarity (M) (pH 10.5)
After incubation, spin down the plate again and Add 30 µL stop solution 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.