May 23, 2022
In Vitro B-galactosidase and B-hexosaminidase Activity Assay (Total Cell Lysate)
- 1Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London (UCL)
Protocol Citation: Laura Smith 2022. In Vitro B-galactosidase and B-hexosaminidase Activity Assay (Total Cell Lysate). protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3p8r7l25/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: April 28, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 61620
Keywords: ASAPCRN
Abstract
Synthetic substrate can be used at acidic pH (pH 4.1) to assess activity of lysosomal hydrolases, β-Galactosidase and β-Hexosaminidase. The substrate is cleaved by β-Galactosidase and β-Hexosaminidase and produces a fluorescent product proportional to activity.
Reagents
Reagents
Buffers: McIlvaine citrate–phosphate (MV), pH 5.4
Substrates:
- β-Galactosidase: 4-Methylumbelliferyl β-D-galactopyranoside (Sigma M1633)
- β-hexosaminidase: 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma M2133):
Standard:4–methylumbelliferone (MWt. 176)
Stopping solution:0.25 M glycine buffer pH 10.4 reagent.
Inhibitors: CBE (Sigma 5424) and DNJ (Enzo BML-SL230-005).
Preparation of reagents
Preparation of reagents
McIlvaine citrate–phosphate (MV):
| A | B | |
| pH 4.1 | ||
| 0.1 M citric acid | 30 ml | |
| 0.2 M Na2HPO4 | 20 ml, then 1:1 |
0.1M citric acid monohydrate (Mwt = 210.14 g/mol) – 5.2535 g in 250 mL dH2O
0.2M Na2HPO4 (Mwt = 141.9 g/mol) – 7.098 g in 250 mL dH2O
Substrates:
Substrates:
β-Galactosidase: 4-Methylumbelliferyl β-D-galactopyranoside (Sigma M1633):
Take 3.4 mg mg in 10 mL dH2O and heat at 80’C until dissolved (0.34mg/ml)
Make up in two bijou tubes; vortex to dissolve or sonnicate and store at -20.
For each experiment, heat at 60-80’C in oven to ensure all powder is solubilised.
β-hexosaminidase: 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma M2133):
Take 27 mg in 10 ml dH2O and heat at 80’C until dissolved (2.71mg/ml)
Make up in two bijou tubes of 5 ml and store at -20’C.
For each experiment, heat at 60-80’C in oven to ensure all powder is solubilised.
Standard: 4–methylumbelliferone (MWt. 176) (Sigma M1381):
Desired concentration is 1 nmol in 200 uL aliquots.
Take 2 mg in 1.5 mL dH2O, then do a 1:1000 dilution in dH2O to give a 0.2ug/200ul concentration.
Aliquot into 200 uls in eppendorfs.
When using in experiment, add 1 mL glycine buffer to Eppendorf and load 200 uL into each well.
Stopping buffer: 0.25 M glycine buffer pH 10.4 reagent:
Make up 64g NaOH in 200 mL dH2O.
Make up 150g glycine in 1600 mL dH2O.
When all mixed, add both together.
Ensure pH is 10.4 and make up to 2 L with dH2O.
Apparatus / Instrumentation
Apparatus / Instrumentation
37oC water bath
Plate reader, excitation 360 nm, emission 460 nm, sensitivity=50
Sample preparation
Sample preparation
Enzyme:
Samples are resuspended in water or 1% (v/v) TX-100 in PBS. For TX-100 lysis, cells incubated on ice for 15 mins and debris/nuclei removed by centrifugation at 17, 000 x g, 10 min, 4 °C. Supernatant containing GBA enzyme placed in fresh tube. All samples are sonicated in water bath for 1 minute. Protein concentration measured with BCA protein assay.
Method Protocol
Method Protocol
Dilute in water a portion of the sonicate to give a protein concentration of 0.25 - 4 mg / ml.
Set up mix in eppendorf tubes for each well as follows:
Make a master mix e.g multiply by number of wells.
B-gal:
20µl B-gal/HEX buffer pH 4.1
10µl B-gal substrate solution
B-hex:
20µl B-gal/HEX buffer pH 4.1
10µl B-gal substrate solution
Add 10 µl diluted enzyme sample in to duplicate wells of a 96 well plate. (Note: may have to optimise volume of sample loaded depending on sensitivity of fluorescence machine used).
Add 10 µl of lysis buffer used (water of TX-100 in PBS) to duplicate wells to serve as substrate blanks.
Add 30 µl of reaction mixture to each well
Incubate at 37°C for 30 minutes.
Add 200 µL stopping solution to each well
Standard:
To 1 nmol 4–methylumbelliferone standard in 200 µl H2O add
1.0 ml stopping reagent.Mix and 200 ml to empty wells to serve as a fluorescence standard.
Read fluorescence, excitation 360 nm, emission 460 nm.
Calculate activity in nmol / hr / mg protein