| Assay Method Information | |
| | 15-PGDH Enzyme Activity Assay |
| Description: | a. A solution of pH 7.5 containing 50 mM Tris-HCl, 0.01% Tween 20 was prepared with ultrapure water as a reaction buffer;b. A 10 mM mother liquor of the compound to be tested was prepared with DMSO, and then the reaction buffer was used to dilute the mother liquor of the compound to be tested to obtain solution 1 of the compound to be tested at a concentration of 40,000 nM, and then the solution 1 of the compound to be tested was serially diluted into solutions 2-9 (or 2-12) of the compound to be tested at 9 (or 11) concentrations with a gradient difference of three-fold. 5 μL of each concentration of solutions of the compound to be tested was respectively taken and added into a 384-well plate as test wells;c. Then 5 μL of the reaction buffer was added to the blank wells of the 384-well plate as positive control and blank control wells, respectively;d. The reaction buffer was used to prepare a 15-PGDH protein solution at a concentration of 5 ng/μL, 5 μL of the 15-PGDH protein solution was taken and added to the test wells and positive control wells, and meanwhile another 5 μL of the reaction buffer was added to the blank control wells, then the plate was centrifuged at 2000 rpm for 30 seconds;e. The reaction buffer was used to prepare 5 mM β-NAD and 2 mM PGF2α, respectively, which were mixed at 1:1 by volume to obtain a substrate mixture, 10 μL of the substrate mixture was taken and added to the test wells, positive control wells and blank control wells to start the reaction;f. The fluorescence signal value (Ex/Em=340/450) of each well was detected continuously by using a multifunctional microplate reader. |
| Affinity data for this assay | |
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