| Assay Method Information | |
| | Human ATX Enzyme Inhibition Assay |
| Description: | ATX is diluted to 2.2 nM in assay buffer. Choline oxidase and horseradish peroxidase are diluted to 7.3 U/ml and 14.7 U/ml, respectively. 18:1 LPC and Amplex Red® are diluted to 110 μM and 183.3 μM, respectively (solution protected from light). 2.2 μL of compound pre-dilution or 50% DMSO is added to the reaction plate followed by 25 μL of ATX or assay buffer (negative control). Assay plate is mixed and incubated at room temperature for 10 mins. 15 μL of choline oxidase/horseradish peroxidase is then added. To initiate the reaction 15 μL of LPC 18:1/Amplex Red is added. Assay plate is mixed and incubated at room temperature in the dark. Fluorescence is measured at 5 mins (for background subtraction) and 90 mins.Final Assay Concentrations: hATX: 1 nM 18:1 LPC: 30 μM Choline Oxidase: 2 U/ml Horse Radish Peroxidase: 4 U/ml Amplex Red®: 50 μM DMSO: 2%Fluorescence at 5-minutes is subtracted from the 90-minute end point data and normalized with respect to the positive control. IC50 values are calculated. |
| Affinity data for this assay | |
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