TITLE: In Vitro B-galactosidase and B-hexosaminidase Activity Assay (Total Cell Lysate)
AUTHORS: Laura  Smith

[DESCRIPTION]
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.

[STEPS]
SECTION: Reagents
1. 
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).

SECTION: Preparation of reagents
2. McIlvaine citrate–phosphate (MV):

 
            GCase   + NaT      GCase      HEX   and β-gal               pH   5.4      pH   4.5      pH   4.1        0.1   M citric acid      44   ml       54   ml      30   ml        0.2   M Na2HPO4      56   ml      46   ml      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
SECTION: Substrates:
3. 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.
SECTION: Substrates:
5. 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.
SECTION: Substrates:
6. 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.
SECTION: Apparatus / Instrumentation
7. 37oC water bath
Plate reader, excitation 360 nm, emission 460 nm, sensitivity=50
SECTION: Sample preparation
8. 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.


SECTION: Method Protocol
9. Dilute in water a portion of the sonicate to give a protein concentration of 0.25 - 4 mg / ml.
SECTION: Method Protocol
10. 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
SECTION: Method Protocol
11. 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).
SECTION: Method Protocol
12. Add 10 µl of lysis buffer used (water of TX-100 in PBS) to duplicate wells to serve as substrate blanks.
SECTION: Method Protocol
13. Add 30 µl of reaction mixture to each well
SECTION: Method Protocol
14. Incubate at 37°C for 30 minutes.
SECTION: Method Protocol
15. Add 200 µL stopping solution to each well 
SECTION: Method Protocol
16. 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.

SECTION: Method Protocol
17. Read fluorescence, excitation 360 nm, emission 460 nm.
SECTION: Method Protocol
18. Calculate activity in nmol / hr / mg protein
SECTION: Substrates:
4. 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.