| Assay Method Information | |
| | In Vitro ELISA (Enzyme-Linked Immunosorbent Assay) of ADAMTS-5 Activity |
| Description: | Table 2: In this assay, the enzymatic activity of recombinant ADAMTS-5 protein (catalog #2198-AD, R&D Systems) was assayed with a protein substrate, the aggrecan IGD protein. The aggrecan IGD protein is a polypeptide connecting human aggrecan globular domains 1 and 2 (T331-G458) expressed in E. Coli with a C-terminal His-tag (catalog #30411000, BIOTEZ). The enzymatic product ARGSVIL-peptide was detected using an ELISA kit from BioTEZ (catalog #30510111). An assay buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl2), 0.1% CHAPS and 5% Glycerol was prepared. Recombinant ADAMTS-5 protein was diluted to 0.3 nM in the assay buffer. Ten μl of buffer and 10 μl of compound solution was transferred to each well of a 96-well plate and incubated at room temperature for 15 minutes. Substrate aggrecan-IGD was diluted to 100 nM with the assay buffer and 20 μl was added to each well. The plate was incubated at 37° C. for 45 minutes. After incubation, the newly generated epitope ARGSVIL-peptides were measured using the Aggrecanase Activity ELISA Assay Kit following the manufacturer's instructions. Then, 100 μl of stop solution was added and the absorbance of each well was read at 450 nM, using 620 nM as reference on a TECAN plate reader. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |