| Assay Method Information | |
| | Inhibition test of compounds on 15-PGDH enzyme |
| Description: | 15-PGDH (R&D Systems, Cat. No.: 5660-DH-010) was prepared to twice the final concentration, i.e., 30 nM, using Assay Buffer (50 mM Tris-HCl, pH 7.5, 0.01vol % Tween 20). The obtained mixture was then added to a 384-well white plate (Cisbio Bioassays, Cat. No. 66PL384025) at 8 μL/well. Negative control wells (with Assay Buffer only and without enzyme) were set. The compound was then prepared to 4 times the final concentration using Assay Buffer, i.e., diluted 3-fold to 10 concentrations starting from 4000 nM. The obtained mixture was added to the above white plate at 4 μL/well, mixed well, centrifuged at 1000 rpm for 1 minute, and incubated at 25° C. for 10 minutes. Both positive control wells (with 15-PGDH only) and negative control wells (without 15-PGDH) were set. A mixture of NAD+(Select, Cat. No. S2518) and PGE2 (R&D Systems, Cat. No.: 2296/10) was then prepared using Assay Buffer. NAD+ and PGE2 were prepared to four times their final concentrations, i.e., 2 mM and 0.12 mM, respectively, using Assay Buffer. The obtained mixture was then added to the above white plate at 4 μL/well, mixed well, centrifuged at 1000 rpm for 1 minute, and incubated at 25° C. for 30 minutes for the reaction. The fluorescence was detected at an excitation wavelength of 340 nm and an emission wavelength of 485 nm using instrument TECAN SPARK 20M. |
| Affinity data for this assay | |
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