Assay Method Information
Assay Name:  In vitro PDE4B Enzyme Activity Detection Assay
Description:  1. Experimental MaterialsName Brand Cat No.PDE4B1 enzyme BPS 60041Trequinsin TOCRIS 2337/10IMAP FP IPP Explorer Kit Molecular Device R8124FAM-cAMP Molecular Device R7506OptiPlate  -384 F PerkinElmer 6007279black assay plate 384 well Echo plate Labcyte PP-02002. Experimental StepsFirst, a stock solution of the compound at a concentration of 10 mM was prepared in a test tube with 90% DMSO (10% water), and it was used to prepare a series of dilutions with a dilution factor of 1:5 and final concentrations starting from 100 μM to as low as 0.05 nM. 0.2 μl of the compound solution was transferred into a 384-well reaction plate, and 0.2 μl of 100% DMSO was transferred into both the negative and positive controls. Then, 10 μl of 2 PDE4B1 enzyme solution (making a final concentration of 0.04 nM) was added to each well, and 10 μl of 1 reaction buffer instead of the enzyme solution was added to the zero enzyme activity control wells. The plate was centrifuged at 1,000 rpm for 1 min and incubated at room temperature for 15 min. Next, 10 μl of 2 FAM-cAMP substrate solution (making a substrate final concentration of 0.1 μM) was added to each well of the 384-well reaction plate. The plate was centrifuged at 1,000 rpm for 1 min and the reaction was carried out at 25 C. for 30 min. After completion of the reaction, 60 μl of reaction stop solution was added to each well of the 384-well reaction plate to terminate the reaction, and the plate was incubated with shaking at 600 rpm on a shaker at room temperature in the dark for 60 min. After the incubation, the RLU data were read and the inhibition rate was calculated. The IC50 value was calculated from the concentration-inhibition fitted curve, wherein the maximum value refers to the reading of the DMSO control and the minimum value refers to the reading of the zero enzyme activity control.
Affinity data for this assay
 
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