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| | Cathepsin L Enzyme Assay |
| Description: | The protease was activated by the addition of dithiothreitol, ethylenediaminetetraacetic acid (EDTA), and sodium acetate followed by incubation at 37°C for 2 h. Proteinase activity was measured fluorometrically using Z-Phe-Arg-NHMec as substrate. The substrate was stored as a 1 mM stock solution in dimethyl sulfoxide (DMSO). Assays were carried out using a final concentration of 10 μM substrate in 0.1 M sodium acetate containing 0.1 M dithiothreitol and 0.5 M EDTA. The enzyme and inhibitor were incubated together for 15 min at 37°C, at which stage the substrate was added. The mixture was incubated at 37°C for 30 min. The reaction was stopped by the addition of 150 μL of a 10% acetic acid solution. The amount of 7-amino-4-methylcoumarin (-NHMec) released was measured using a PerkinElmer fluorescence spectrophotometer with excitation set at 370 nm and emission at 440 nm. |
| Affinity data for this assay | |
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