| Assay Method Information | |
| | In Vitro Fluorescence Assay of ADAMTS-4 or ADAMTS-5 Activity |
| Description: | Table 1: A FRET (fluorescence resonance energy transfer) peptide was cleaved by recombinant ADAMTS-4 or ADAMTS-5 proteins into two separate fragments resulting in an increase of fluorescence signal which was quantified. The peptide was 5-FAM-TEGEARGSVILLK(5-TAMRA)K-NH2, customized from ANASPEC. ADAMTS-4 recombinant protein (catalog #4307-AD) and ADAMTS-5 recombinant protein (catalog #2198-AD) were purchased from R&D Systems. An assay buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS and 5% Glycerol was prepared. A volume of 2.5 μl of compound in the assay buffer was dispensed to a 384-well plate, and 2.5 μl of ADAMTS-4 or ADAMTS-5 protein (final concentration in the reaction was 10 nM) was added. The compounds and proteins were pre-incubated at room temperature for 15 minutes. Then, 5 μl of substrate was added to each well. The final substrate concentrations for ADAMTS-4 and ADAMTS-5 were 15 μM and 8 μM, respectively. The fluorescence signal in each well was determined, after incubation at 37° C. for 3 hours, on a TECAN plate reader (Excitation, 490 nm; Emission, 520 nm). |
| Affinity data for this assay | |
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