Bioactivity |
Activity of ab134430 is determined with recombinant aggrecan interglobular domain. ab134430 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 0.2-3.0 nM ADAMTS1 in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 µM leupeptin, 1 µM pepstatin, 1 mM Pefabloc, 0.05 % Brij 35 for 15 min at 37°C. 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 to the neoepitope. Under the specified conditions the hydrolysis rate is > 0.06 nM hydrolysed substrate/min x nM truncated ADAMTS1. When related to mg enzyme, the value is > 1.4 nmoles hydrolyzed substrate/min x mg ADAMTS1.
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