| Assay Method Information | |
| | ATX Enzyme Activity Inhibition Assay |
| Description: | The tested compounds were dissolved with dimethyl sulfoxide (DMSO) to prepare 20 mM of stock solutions, the stock solutions were subjected to 4-fold gradient dilutions with DMSO, with the initial concentration of 20 µM and a total of 8 concentration gradients. 20 nL of diluted compound was transferred to a 384-well plate using Echo 550 and 5 µL of ATX enzyme solution formulated with 4 × Tris-HCl reaction buffer to the well, then the 384-well plate was centrifuged at 1000 rpm for 1 min and pre-incubated for 1 min and pre-incubated at room temperature for 15 min. Subsequently, 5 µL of 16:0-LPC foumulated with the above 4 × reaction buffer and 10 µL of assay solution containng 2 × Amplex UltraRed reagent, choline oxidase and horseradish peroxidase (HRP) foumulated with the same reaction buffer were added to each well, then the 384-well plate was centrifugated at 1000 rpm for 1 min. The fluorescence signals were read at an excitation wavelength of 530 nm and an emission wavelength of 590 nm by Synergy 2 microplate reader. The inhibition rate of the compound against enzyme reaction was calculated according to the fluorescence intensity ratio, and IC50 of the compound was analysed and calculated by GraphPad Prism 5 software. |
| Affinity data for this assay | |
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