| Assay Method Information | |
| | fluorescent peptide assay |
| Description: | The assays were carried out with 20 nM enzyme (except for MPI3, for which 10 nM enzyme was used) and 10 μM substrate at 37oC with continuous shaking. All the analyses were carried out in triplicate. The substrate (DABCYL-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS) was purchased from Bachem and stored as 1 mM solution in 100% DMSO. Enzyme activity was monitored by fluorescence with excitation at 336 nm and emission at 455 nm wavelength. The dilution buffer (used for enzyme and substrate dilution) is 10 mMNaxHyPO4,10 mM NaCl, 0.5 mM EDTA, pH 7.6. Final composition of the assay buffer is 10 mM NaxHyPO4, 10 mM NaCl, 0.5 mM EDTA, 2 μM DTT (coming from enzyme stock solution), pH 7.6 with 1.25% DMSO. All the inhibitors were stored as 10 mMin 100%DMSO solutions in -20 oC freezer. |
| Affinity data for this assay | |
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