Sep 12, 2024
GCase activity in mouse brain samples
- 1German Center for Neurodegenerative Diseases (DZNE)
Protocol Citation: Pietro La Vitola 2024. GCase activity in mouse brain samples. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8eq7v5b/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: September 12, 2024
Last Modified: September 12, 2024
Protocol Integer ID: 107459
Keywords: GCase activity, mouse, brain
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for assessing the beta-glucocerebrosidase activity in fresh collected mouse brain samples
Materials
Reagent Buffer (pH 4.10):
Citrate phosphate buffer (0.15M) (Sigma C0909-500g H2O free MW258,06g/mol)
ddH2O
Stopping buffer (pH 10.4):
Glycine buffer (0.25M) (Roth 3908.2 MW 75,07g/mol)
ddH2O
Substrate :buffer:
Sodium taurocholate (Sigma 86339, 150mM)
4-Methylumbelliferyl β-D-glucopyranoside (MUG, Sigma M3633, 10mM)
ddH2O
Standard:
4-Methylumbelliferone (M1381, Sigma)
Methanol
Samples homogenization
Samples homogenization
10m
10m
Homogenize fresh collected mouse brain samples in lysis buffer (50 mM Tris-HCl, pH 7.4 and 750 mM NaCl, 5 mM EDTA and 10%Triton X-100).
Centrifuge samples at 5000 g (00:10:00 at 4 °C )
10m
Collect the supernatant
Measure protein concentration with the Pierce BCA Protein Assay Kit (Fisher Scientific, 23225).
Assesment of GCase activity
Assesment of GCase activity
1h
1h
Make a serial dilution of 4-Methylumbelliferone (M1381, Sigma) dissolved in 1% Methanol ddH20
Put 10µL of each standard dilution and of each sample in a pre-prepared black 1.5 ml tube (in duplicate or triplicate).
Add 25µL of Reaction Buffer and 65µL of Substrate buffer in each tube
Incubate01:00:00 at 37 °C
1h
Remove samples from the incubator and add 90µL of Stopping buffer to each tube
Load 150µL of all samples, standards and blank (35µL of Reaction Buffer + 65µL of Substrate buffer + 90µL of Stopping buffer) into a 96 well black plate
Measure fluorescence using plate reader (Excitation at 365 nm and emission at 450 nm)
Calculate the amount of 4-Methylumbelliferone generated in each sample based on the standard curve
Normalize the data obtained based on the amount of protein (mg) that were in each reaction tube