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| | Enzymatic Chymase Assay |
| Description: | The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 ul of a 50-fold concentrated solution of test substance in DMSO, 24 ul of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 ul of substrate solution (final concentration 10 uM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtitre plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Mainnedorf, Switzerland). One test compound is tested on the same microtitre plate in 10 different concentrations from 30 uM to 1 nM in a double determination. The data are normalized (enzyme reaction without inhibitor=0% inhibition, all assay components without enzyme=100% inhibition) and IC50 values are calculated using in-house software. |
| Affinity data for this assay | |
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