| Assay Method Information | |
| | Cathepsin Activity Assay |
| Description: | A microplate-based screening procedure was used to study the effect of inhibition of re-Cat S, re-Cat L and re-Cat K on several compounds. The assays were performed in quadruplicates at 25 °C in a buffer containing 50 mM MES, pH 6.5, 2.5 mM EDTA and 1 mM DTT for re-Cat S, or 100 mM NaOAc, pH 5.5, 1 mM DTT and 2 mM EDTA for re-Cat L, or 50 mM MES, pH 5.5, 5 mM DTT and 2.5 mM EDTA for re-Cat K. Each of tested compounds at 100 nM was incubated with 50 nM re-Cat S, 31.25 pM re-Cat L or 10 nM re-Cat K at 37 °C for 10 min before initiating the reaction with the addition of 5 μM relevant substrate (Z-Phe-Arg-AMC for Cat L and Cat K, and Z-Val-Val-Arg-AMC for Cat S), respectively [Ward et al., J. Med. Chem., 45:5471-82]. Reactions in microplates were kept at 37 °C for another 30 min and then to collect the fluorescent signals from each well in microplates. E64 and 1% DMSO were applied as positive and solvent control, respectively. Fluorimetric assays were performed at 37 °C on a Perkin Elmer LS55 fluorescence spectrophotometer (Waltham, MA) with excitation at 370 nm and emission at 460 nm wavelengths. The inhibitory ability of a tested compound was determined by decreased fluorescence compared to DMSO control. |
| Affinity data for this assay | |
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