| Assay Method Information | |
| | Effect of Compounds on PDE4B1 Activity Assay |
| Description: | The effect of compounds on PDE4B1 activity was detected using a fluorescence polarization kit (IMAP FP IPP Explorer KIT, Molecular Device, Cat #R8124). The IMAP Reaction Buffer containing 0.1% BSA (5×) in the kit was diluted into a 1-fold reaction buffer containing 1 mM DTT. 0.12 nM PDE4B1 (BPS, Cat #60041) was added to the 1-fold reaction buffer to form a 2-fold enzyme solution. 10 μL of the 2-fold enzyme solution was added to wells of a 384-well reaction plate. For the negative control well, 10 μL of the 1-fold reaction buffer was used instead of the enzyme solution. Centrifugation at 1000 rpm for 1 minute and incubation at room temperature for 15 minutes were carried out. 0.2 uM FAM-labeled cAMP (FAM-cAMP, Molecular Device, Cat #R7506) was added to the 1-fold reaction buffer to form a 2-fold substrate solution. 10 μL of the 2-fold substrate solution was added to each well of the 384-well reaction plate. Centrifugation at 1000 rpm for 1 minute was carried out. A reaction was carried out at room temperature for 20 minutes. IMAP Progressive Binding Buffer A (5×), IMAP Progressive Binding Buffer B (5×), and IMAP Progressive Binding Reagent (provided by IMAP FP IPP Explorer Kit) were prepared into a reaction stop solution according to the instructions for use. 60 μL of an 80/60-fold reaction stop solution was added to each well of the 384-well reaction plate to stop the reaction, and the reaction product was centrifuged at 1000 rpm for 1 minute. Incubation at room temperature in the dark for 60 minutes was carried out. The mP values (Ex480/Em535 (s), Em535 (p)) were read by a microplate reader (Envision). |
| Affinity data for this assay | |
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