Table 1 |
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Assay conditions for each enzyme activity assay. |
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|
ENZYME |
Punch diameter |
Reaction buffer |
Fluorogenic substrate |
Additional reagents |
Incubation time |
Stop buffer |
|
|
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|
alpha-galactosidase A |
1.5 mm |
30 μL citrate-phosphate 0.3 M pH 5.0 |
30 + 30* μL 4 MU-alpha-D-galactopyranoside 4.2 mM |
10 μL N-acetyl-galactosamine 0.25 M |
20 hours |
10% ethylenediamine 1.32 M pH 11.3 |
|
|
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|
alpha-iduronidase |
1.5 mm |
10 μL sodium formate 0.2 M pH 2.8 |
20 + 20* μL 4MU-alpha-L-idopyranoside 2 mM |
10 μL D-saccharic acid 1,4-lactone monohydrate 3 mM |
20 hours |
10% ethylenediamine 1.32 M pH 11.3 |
|
|
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|
beta-glucosidase |
3.0 mm |
30 μL citrate-phosphate 0.4 M pH 5.2 |
50 + 50* μL 4MU-beta-D-glycopyranoside 20 mM |
40 μL sodium taurodeoxicolate hydrate 0.75% |
20 hours |
10% ethylenediamine 1.32 M pH 11.3 |
|
|
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|
alpha-glucosidase |
1.5 mm |
20 μL sodium acetate 0.4 M pH 4.0 and 6.5 |
20 + 20* μL 4MU-alpha-D-glycopyranoside 2.8 mM |
40 μL acarbose 40 μM |
24 hours |
10% ethylenediamine 1.32 M pH 11.3 |
|
|
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|
beta-galactosidase |
1.5 mm |
20 μL citrate-phosphate 0.1 M pH 4.4 |
20 + 40* μL 4MU-beta-D-galactopyranoside 0.8 mM |
20 μL sodium chloride 0.9% |
3 hours |
10% ethylenediamine 1.32 M pH 11.3 |
|
|
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|
* addition in blank wells after incubation MU: methylumbilliferyl |
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|
Müller et al. Diagnostic Pathology 2010 5:65 doi:10.1186/1746-1596-5-65 |
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