| Assay Method Information | |
| | Inhibitory Activity Assay |
| Description: | Necessary amounts of test compounds were weighed, and 100% dimethylsulfoxide (DMSO) was added thereto to adjust the concentration to 10 mM. The solutions were stored in a freezer as stock solutions of each test compound. After being thawed when required, the solutions were diluted 20-fold with 100% DMSO to give a 500 μM concentration. Further, 10-fold serial dilutions were made with 100% DMSO to prepare test compounds of different concentrations. 2 μl of solutions containing one of each of the test compound were separately added into 1.2 ml tubes in which 23 μl of 20 mM Tris-HCl (pH 7.4) had been placed beforehand. 25 μl of a PDE4D enzyme solution diluted at an optimal ratio determined in (3) above were added on ice to each of the tubes, and 50 μl of a substrate solution containing 2 μM[3H] cAMP prepared by dilution with a 20 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2 was added thereto. The final DMSO concentration in the reaction liquid was 2%. After mixing, the mixture was incubated for 10 minutes at 30° C. At the completion of the incubation, the tubes were placed in a bath containing boiling water for 3 minutes, and the reaction was stopped. After cooling the tubes in ice, 25 μl solution of 0.2 mg/ml snake venom was added thereto, and after mixing the mixture was incubated for 10 minutes at 30° C. At the completion of the incubation, 0.4 ml of a Dowex 1×8 resin solution prepared in an EtOH:H2O (1:1) solution was added thereto. After mixing, the tubes were allowed to stand at room temperature for at least an hour. 50 μl of the supernatant in one of each of the tubes was moved to one of the wells of a topcount plate, and the plate was dried overnight. 3H radioactivity (cpm) was measured using a TopCount. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |