Bioactivity |
Aggrecanase activity of ab134432 is determined with recombinant aggrecan interglobular domain. ADAMTS4 hydrolyzes the aggrecanase site within this domain (peptide bond E 373 -A 374 in Human aggrecan). The recombinant substrate is incubated at a concentration of 0.1 µM with ADAMTS5 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 M CaCl2, 1 µM leupeptin, 1 µM pepstatin, 1 mM Pefabloc, 0.05 % Brij 35 for 15 min at 37°C. A series of ADAMTS5 dilutions ranging from 103 - to 105 -fold is tested. Substrate cleavage at the aggrecanase-site is estimated from the appearance of the hydrolysis fragment with the novel N terminus ARGSVIL. The fragment is quantified with two monoclonal antibodies, one directed against the neoepitope ARGSVIL, the other against the sequence C terminal from the neoepitope. Under the specified conditions the hydrolysis rate is > 0.5 nmoles hydrolyzed substrate/ min x ml ADAMTS5 preparation or > 5 nmoles hydrolyzed substrate/ min x mg.
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