| Assay Method Information | |
| | Assay for Inhibitory Activity Against PDE4B1 at Enzymological Level |
| Description: | Procedures:1. 20 μM FAM-Cyclic-3′,5′-AMP was 100-fold diluted to 200 nM with a PDE assay buffer. The unused portion of the stock solution should be aliquoted and stored at −20° C.2. 1 M DTT was added to the 200 nM FAM-Cyclic-3′,5′-AMP dilution in a ratio of 1:500.3. The FAM-Cyclic-3′,5′-AMP dilution was added to a test plate at 25 μL/well, except for the blank control wells (Blank).4. The PDE assay buffer was added to the blank control wells (Blank) at 45 μL/well, and the PDE assay buffer was added to the substrate control wells at 20 μL/well.5. Working solutions of the compounds were added to the corresponding wells of the test plate at 5 μL/well. The PDE assay buffer (10% DMSO) was added to the positive control wells, the substrate control wells, and the blank control wells (Blank) at 5 μL/well.6. The PDE4B1 enzyme was thawed on ice in advance, and the completely thawed enzyme was diluted to 1 pg/μL with the PDE assay buffer. The enzyme solution was added to the compound wells and the positive control wells at 20 μL/well. The unused portion of the enzyme stock solution should be aliquoted and stored at −80° C.7. The test plate was incubated at room temperature for 1 hour with a compound concentration of 10 μM and a DMSO concentration of 1%.8. The binding agent was diluted with the binding agent diluent (cAMP) in a ratio of 1:100 for later use.9. After the incubation, the binding agent dilution was added to the test plate at 100 L/well, and the plate was incubated at room temperature for 30 min with slow shaking.10. After the incubation, an FP assay was performed using Envision with an excitation wavelength of 480 nm and an emission wavelength of 535 nm.11. The IC50 of the compounds was calculated. |
| Affinity data for this assay | |
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