| Assay Method Information | |
| | Enzyme Assay |
| Description: | To 2.5 μl of supernatant (in 96-well plates) was added 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, no Triton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeled substrate, β-glucopyranoside, or a labeled negative controls (α-glucopyranoside or α-galacatopyranoside). Plates were incubated at 37° for 1 hour, followed by the addition of 70 μl stop buffer (0.4 M glycine-NaOH, pH 10.6). Activity of GCase was determined by measuring the emission at 460 nm by exciting at 355 nm using a 1 second read time per well (Victor2 multilabel counter-Wallac) Enzyme activity was normalized to the amount in μl of lysate added, and enzyme activity per μl of lysate was estimated. |
| Affinity data for this assay | |
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