Assay Method Information

Assay Name:  Inhibition Assay
Description:  The TACE inhibition test was carried out by measuring TACE activity in the presence and the absence of the test substance using the thus-obtained TACE as an enzyme, and a fluorescent synthetic substrate Nma(N-methylanthranilic acid)-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Dnp(dinitrophenyl))-D-Arg-NH2 (SEQ ID NO: 1) including the TACE-cleaved sequence of a membrane-bound TNF as a substrate. The TACE inhibition test method is shown below. Namely, 90 μL of an enzyme solution prepared with an assay buffer A (50 mM tris-hydrochloric acid buffer (pH 7.5) including 200 mM sodium chloride, 5 mM calcium chloride, 10 μM zinc sulfate, and 2 mg/mL bovine serum albumin) and 90 μL of a fluorescent synthetic substrate prepared with an assay buffer B (50 mM tris-hydrochloric acid buffer (pH 7.5) including 200 mM sodium chloride, 5 mM calcium chloride, 10 μM zinc sulfate, and 0.05% PLURONIC F-68) were mixed together, and reacted at 37° C. for 1.5 hours. Enzyme activity was then determined by measuring with a fluorescence intensity meter (Labsystems, Fluoroskan Ascent) at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm.
Affinity data for this assay
 

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