| Assay Method Information | |
| | Choline Release Assasy |
| Description: | The purpose of this assay is to detect autotaxin inhibition using a choline release assay.Test compound (10 mM stocks in 100% DMSO) is serially diluted in 100% DMSO resulting in 10 concentrations of 100× inhibitor in half area 96 well plates (Corning 3992). Each of these 10 wells in 100% DMSO is diluted 1:33.33 in assay buffer in round bottom 96 well plates (Fisher 12565502) resulting in 3× concentrations in well containing 3% DMSO. The assay buffer is 50 mM Tris pH8.0, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% TRITON X-100 (Sigma T9284) and 0.01% fatty acid free bovine serum albumin (Sigma A8806). A 20 μl aliquot of each 3× test compound is then added to black flat bottom 96 well plates (Corning 3991) in singlicate. A 20 μl aliquot per well of 3× recombinant human autotaxin, (Echelon, E-4000) (full length human autotaxin with a C-terminal His tag transfected into 293E cells and purified via nickel chelate and size exclusion chromatography) is then added to every well except for the no enzyme control wells. A 20 μl aliquot per well of assay buffer is added to the no enzyme control wells. A 20 μl aliquot of a 3× cocktail containing choline oxidase (Sigma C5896), horseradish peroxidase (Sigma P8125), amplex ultrared (Invitrogen A36006) and the autotaxin substrate lysophosphatidylcholine (LPC) 16:0 (Avanti Polar Lipids 855675P) is added to each well while avoiding exposure to light. The final concentrations in the well of choline oxidase, horseradish peroxidase, amplex ultrared and LPC 16:0 are 0.4 units/ml, 4 units/ml, 40 μM and 30 μM respectively. The plate is then sealed with aluminum foil seals and incubated at 37° C. for 1 hour in a Labline Imperial III incubator. During this incubation, LPC is cleaved by autotaxin resulting in Lysophosphatidic Acid (LPA) 16:0 and choline. The choline that is released is oxidized by choline oxidase resulting in betaine and hydrogen peroxide. The hydrogen peroxide reacts with the horseradish peroxide and amplex ultrared to form the fluorescent molecule resorufin. |
| Affinity data for this assay | |
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