| Assay Method Information | |
| | DPP-IV Activity Inhibition Tests In Vitro |
| Description: | DPP-IV could hydrolyze Gly-Pro-Aminoluciferin at room temperature to generate Aminoluciferin, which could produce glow type luminescent signals in a luciferase reaction system provided by a DPPIV-Glo protease test kit, and the strength of the luminescent signals was in direct proportion to the enzyme activity of DPP-IV.1. Experimental purposes:to evaluate the inhibition effects of compounds I-1 I-4 in the present invention by observing their activity inhibition to DPP-IV enzyme.2. Experimental materials:2.1 humanized recombinant DPP-IV: SIGMA product, article number D3446-10UG.2.2 DPPIV-Glo protease detection kit: Promega product, article number G8351.2.3 Trizma base: Sigma product, article number T6066-1KG: prepared into 10 mM Tris-HCl, pH 8.0.2.4 384 OptiPlate: PerkinElmer product, article number 6007299.2.5 Liquid treatment instrument: Bravo (Agilent company); Echo (Labcyte company).2.6 Detection instrument Envision (PerkinElmer company).3. Experimental methods:3.1 Diluting tested samples in a gradient dilution to ten concentrations by DMSO with Bravo, and then transferring 250 nl of samples to 384 OptiPlate with Echo.3.2 Diluting dipeptidyl peptidase IV (Sigma) to 0.2 ng/ml solution with 10 mM Tris-HCl (pH 8.0), adding the samples to be detected in, per well 25 μl. Meanwhile, a blank control (including substrate but no enzyme and samples) and positive control (including substrate and enzyme but no samples) were also set up.3.3 Adding 25 μl of DPPIV-Glo Reagent (prepared according to instructions in DPPIV-Glo protease detection kit, containing 20 μM DPP-IV substrate Gly-Pro-Aminofluorescein and luciferase reaction system) into each well.3.4 Reacting at room temperature for 60 min, determining the luminescence intensity by Envision.3.5 Calculating the enzyme activity of DPP-IV according to the luminescence intensity, enzyme activity=(sample luminescence intensity values−blank control luminescence intensity values/(positive control luminescence intensity values−blank control luminescence intensity values)×100.3.6 Calculating IC50 of the samples according to the enzyme activity using GraphPad Prism5.0 software. |
| Affinity data for this assay | |
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