| Assay Method Information | |
| | Golgi α-Mannosidase II (alpha-hGMII) and α-Mannosidase (alpha-hLM) Activity Assay |
| Description: | Compounds were diluted to give the final concentration of 100 μM and mixed with 4-MU-α-D-mannopyranoside as substrate and human Golgi α-mannosidase II in phosphate buffer (0.1 M Sodium phosphate dibasic, pH 7.0) or human lysosomal α-mannosidase in citric phosphate buffer (0.1 M Sodium phosphate monobasic monohydrate, 0.5 mM citric acid monohydrate, pH 4.6). The assay was carried out at 37° C. for a certain time (1 h for α-hLM, 2 h for α-hGMII). Stop solution (0.5 M K2CO3(aq), pH 10.8) was then added to the reactions and the fluorescence was determined at 355 nm excitation and 460 nm emission (SpectraMax M5, Molecular Devices). Inhibition was performed as relative enzyme activity to control. The active compounds were selected for libraries preparation and further tested at lower concentration to determine their IC50 values. The assays performed in 384-wells of the microtiter plates. |
| Affinity data for this assay | |
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