| Assay Method Information | |
| | In Vitro Inhibitory Assay |
| Description: | 1) Preparation of test compound plate: The test compounds and positive control AZD7986 were dissolved in 100% DMSO to obtain compound stock solutions with a final concentration of 10 mM. The compound was diluted with DMSO to a 384-well echo plate at a concentration of 100× of the highest concentration at the beginning of the experiment. 0.2 μL of the diluted compound was pipetted to a black-bottomed 384-well reaction plate for later use. The negative control well was DMSO.2) Activation of rhCatC: RhCatC and rhCatL were added to the activation buffer to obtain a final concentration of rhCatC of 100 μg/mL and a final concentration of rhCatL of 20 μg/mL, and incubated at room temperature for 1 hour.3) Enzyme activity reaction: Activated rhCatC was diluted to 0.4 μg/mL with the reaction buffer. The solution was added to the black-bottom 384-well reaction plate (10 L per well). 10 μL of the buffer was added for the vehicle control group. The plate containing compounds in its wells was incubated at room temperature for 30 minutes. H-Gly-Arg-AMC was diluted to 97 μM with reaction buffer, and 10 μL of the solution was added to each well.4) Fluorescence detection: The plate was read with Envision, and the fluorescence intensity was measured at Ex 355 nM and Em 460 nM.5) Calculation of inhibition rate and IC50: Inhibition rate: formula (1): inhibition rate %=(maximum value−signal value)/(maximum value−minimum value)×100. |
| Affinity data for this assay | |
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