Tromethamine hydrochloride → Citrate

Participants

Reaction Steps

A compound solution (2 μL) diluted with an assay buffer containing 50 mM Tris-HCl (pH 7.5), 0.5% TWEEN20, and Complete to 15% DMSO and 90 μg/mL human GOAT-expressing microsome fraction (2 μL) were incubated at room temperature for 20 min, after which a substrate mixed solution (2 μL) containing 30 μM octanoyl-CoA (CHEM-IMPLEX) and 3 μM Ghrelin-biotin (Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg-epsilon aminocaproic acid-biotin (Greiner Coutom synthesis)) was added to 384 white small volume plate (Greiner). After reaction for 20 min, 2 μL of citrate buffer (pH3) (mixed solution of 0.1 M citric acid (39.8 mL) and 0.2 M Na2HPO4 (10.2 mL)), 2 μL of 25 nM anti-active ghrelin antibody (produced in mouse), 5 μL of Eu(K)-Anti Mouse IgG Pab (Cis Bio) 100-fold diluted with HTRF detection buffer (Cis Bio) and 5 μL of streptavidin-Xlent (Cis Bio) 66.6-fold diluted with HTRF detection buffer (Cis Bio) were added. After incubation at room temperature for 1 hr or longer, the fluorescence values at 620 nm and 665 nm were measured by Envision (PerkinElmer). The data was calculated [(665 nm signal/620 nm signal)×104], and the inhibitory activity against human GOAT was shown by the data where 0% control was without compound, and 100% control was without enzyme. The results are shown in Table 34.

Provenance & Evidence