| Assay Method Information | |
| | IC50 Determination |
| Description: | Experiments to determine IC50 values against β5i and β5c for compounds were carried out in 96-well plates. In brief, 1 μL of compound in a 3× series dilution in DMSO at concentration ranging from 100 μM-0.0017 μM were spotted to the bottom of a black 96-well plate with solid bottom. 100 μL of reaction buffer (20 mM HEPES, 0.5 mM EDTA, pH7.5, 0.1% BSA) containing enzyme (final concentration was 0.2 nM for c-20S, and 0.4 nM for i-20S) and substrate (25 μM for suc-LLVY-AMC for β5c and 15 μM for Ac-ANW-AMC) were dispensed into each well, and the plate was then spun at 1000× rpm for 1 minute and then shaked on a shaker for 1 minute. Time course of the hydrolysis of each well was followed by recording the fluorescence of product AMC (Ex 360 nm and Em 460 nm) on a SpectraMax M5 plate reader for 1.5-2 hours. Initial reaction velocity of each well was fit to a dose-dependent inhibition equation using PRISM to determine the IC50. IC50s were determined only for β5i and β5c (Table 3). SDS was used as activator for both enzymes at concentration 0.0200. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |