| Assay Method Information | |
| | TACE Assay |
| Description: | The TACE assay was carried out in 25 mM Tris-HCl buffer (pH 9, 2.5 μM ZnCl2 and 0.005% Brij-35). Then, 15ng TACE was added to buffer solution containing 140μMTACE peptide substrate at a final volume of 50 μL. After incubation for 15 min at 37°C, the reaction was stopped with 25 μL of 1% TFA. An aliquot of 25μL of internal standard solution was then added and the solution mixture was analysed by HPLC. GM6001 and inhibitor A were evaluated under the standard assay conditions. The 15ng TACE was added to 30 μL of 25mM tris-HCl buffer (pH 9, 2.5 μM ZnCl2, 0.005% Brij-35) containing varying concentrations of GM6001 (0, 20, 80, 120, 200, 300, 600, 1500, 2500 nM) or inhibitor A (0, 15, 45, 90, 120, 165, 300, 600, 1200 nM). After incubation for 10 min at 37°C, 2μL of the 3.5nM TACE substrate was added. The final volume was adjusted to 50 μL and the final concentration of the peptide substrate was 140 μM. |
| Affinity data for this assay | |
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