| Assay Method Information | |
| | Biological Assay |
| Description: | Recombinant human procathepsin K was obtained from Enzo Life Sciences. Activation of the proenzyme was performed in 32.5 mM sodium acetate pH 3.5, EDTA 1 mM, NaCl 500 mM, human procathepsin K 5.5 uM, at room temperature. Activation times were optimized and varied between 35 and 150 minutes. Cathepsin K was preincubated with test compounds at various concentrations for 5 minutes at 25C. The assay was initiated by addition of substrate Z-Phe-Arg-aminomethylcoumarin ("Z-F-R-AMC" Bacchem) and the final assay conditions were 1.5 nM cathepsin K, 50 uM Z-F-R-AMC, 150 mM sodium acetate pH 5.5, 2.5 mM EDTA (Omnipure), 2.5 mM DTT (EMD), 0.01% BRIJ 35 (Sigma), and 4.0% DMSO (Acros). Test compounds were serially diluted with DMSO and water to include a final concentration range of 10 uM to 10 pM. The reaction was monitored fluorometrically for 5 minutes at 25C. using black 96-well Corning 3686 assay microplates with a Thermo Fluoroskan Ascent FL microplate reader. |
| Affinity data for this assay | |
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