Assay Method Information
Assay Name:  rhDPP4 inhibitor screening experiment
Description:  The inhibitory activity of the compounds on recombinant human dipeptidyl peptidase 4 (rhDPP4) was evaluated by measuring the median inhibitory concentration (IC50) value of the compounds. In this experiment, rhDPP4 was used to catalyze a substrate to produce luciferin, and reacted with luciferase to produce light signals, wherein the substrate is the luminescent precursor glycine-proline-aminofluorescein (Gly-Pro-aminoluciferin). The intensity of the light signal is directly proportional to the enzyme activity.2. Experimental Method1) 250 nL of gradient diluted compound (4 times dilution, 10 detection concentrations) was transferred to a 384-well plate (PerkingElmer-6007299) using a non-contact nano-upgraded acoustic pipetting system (ECHO). The concentration of dimethylsulfoxide (DMSO) concentration was 0.5% in the final reaction system. Blank control wells (containing DMSO, substrate and 10 mM of tris(hydroxymethyl)aminomethane hydrochlorid (Tris-HCl)) and positive control wells (containing DMSO, substrate and rhDPP4) were set.2) The pre-packed frozen buffer containing luciferase was taken out and restored to room temperature, and then added with substrate to prepare a working solution with a substrate concentration of 20 μM. The rhDPP4 was prepared as a 0.2 ng/mL working solution with 10 mM Tris-HCl (pH8.0) aqueous solution.3) 25 μL of working solution containing 20 μM substrate and 25 μL of working solution containing 0.2 ng/mL rhDPP4 were added to the 384-well plate with the compound added already. The plate was centrifuged at 1000 rpm for 30 s. The plate was sealed with aluminum foil sealing film and incubated at room temperature for 1 hour.4) The light signal intensity was detected with a multi-function enzyme label detector EnVision. The raw data was used to calculate the inhibition activity of the compounds on rhDPP4.
Affinity data for this assay
 
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