| Assay Method Information | |
| | Assay for ADAMTS-7 Enzymatic Activity and Testing of Inhibitory Compounds |
| Description: | Purified recombinant ADAMTS-7 (as of SEQ ID No. 01 or SEQ ID No. 02) was diluted in reaction buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 5 mM CaCl2, 0.004% Brij, 10 μM ZnCl2) fora concentration of approximately 20 nM. 25 μl of the solution were transferred into each well of a 384-well white microtiter plate (Greiner Bio-One 781075) and 1 μl test compound solution (modulator/inhibitor dissolved in DMSO, at the corresponding concentration) or pure DMSO as a control were added per well. The enzymatic reaction was initiated by addition of 25 μl of a 1 μM solution of the FRET substrate, HiLyteFluor-488 DELSSMVLELRGLRT-K(QXL520)-E-NH2; (SEQ ID No. 11, custom synthesis by Anaspec) in the reaction buffer. Amino acids DELSSMVLELRGLRT are derived from Thrombospondin-1 sequence (275-289). An additional carboxyl glutamic acid was added after the QXL520 quencher to increase substrate solubility. The microtiter plate was incubated for 120 min at the temperature of 32° C. The increase of fluorescence intensity was measured in appropriate fluorescence plate reader (e.g. TECAN Ultra) using excitation wavelength of 485 nm and emission wavelength of 520 nm. IC50 values were calculated from percentage of inhibition of ADAMTS-7 activity as a function of test compound concentration. IC50 values derived using functional ADAMTS-7 according to SEQ ID No. 01 or SEQ ID No. 02, respectively, were not distinguishable, both laying within the experimental error. |
| Affinity data for this assay | |
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