PDE4D2 TR-FRET Assay Kit
The PDE4D2 TR-FRET Assay Kit is designed to provide fast and easy identification of inhibitors of PDE4D2 (phosphodiesterase 4D2) using Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET). The PDE4D2 TR-FRET Assay Kit comes in a convenient 96-well format, with enough recombinant purified PDE4D2 enzyme, fluorescently labeled PDE substrate (cAMP), binding agent, and PDE assay buffer for 96 enzyme reactions.
Figure 1: Illustration of the assay principle.
The reaction uses a fluorescein-conjugated (FAM) cyclic monophosphate nucleotide. Phosphodiesterase PDE4D2 catalyzes the hydrolysis of the phosphodiester bond in the cyclic monophosphate nucleotide, releasing the phosphate group for binding. The phosphate group binds to a “Binding Agent” (BA) that is recognized by terbium-labeled donor beads. This results in energy transfer from the terbium to FAM, which emits a fluorescent signal at 520 nm. If unbound to the phosphate group, the terbium-labeled beads emit at λ=490 nm. The fluorescent intensity is measured using a fluorescence plate reader capable of TR-FRET reading and an increase in 520 nm corresponds directly to the activity of PDE4D2. In this assay, the intensity of the signal at 520 nm is directly proportional to PDE4D2 enzymatic activity.
Need us to run inhibitor screens or profile your compounds against PDE4D2? Check out our Phosphodiesterase Screening Services.
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
- Fluorescent microplate reader capable of measuring fluorescence polarization (λex=330 (20 nm bandwidth) and detection at λem= 520 (10 nm bandwidth)
| Catalog # | Name | Amount | Storage |
| 60048 | PDE4D2, GST-Tag* | 1 µg | -80°C |
| 60200 | FAM-Cyclic-3’ , 5’AMP** | 50 µl | -80°C |
| 60393 | PDE Assay Buffer (Incomplete) | 25 ml | -20°C |
| 60394 | Tb donor | 50 µl | -80°C |
| 60390 | PDE Binding Agent | 200 µl | +4°C |
| 78422 | Binding Buffer A | 20 ml | +4°C |
| 78423 | Binding Buffer B | 20 ml | +4°C |
| 0.5 M DTT | 200 µl | -20°C | |
| 79685 | Low binding, black 96-well plate | 1 | Room temp. |
*The concentration of the protein is lot-specific and will be indicated on the tube.
** FAM-Cyclic-3′, 5′-AMP is provided as a powder. Vial will need to be resuspended in 600 µl of Complete PDE Assay Buffer before use.
Phosphodiesterases (PDEs) play an important role in the dynamic regulation of the second messengers cAMP (cyclic adenosine monophosphate) and cGMP (cyclic guanosine monophosphate) signaling, by hydrolyzing them. The PDE superfamily is composed of 11 families, with PDE4, 7 and 8 being cAMP-specific hydrolases, and thus regulating positive and negative responses to it. PDE4 is the second most abundant PDE in cardiovascular tissues. PDE4, is a cAMP-specific PDE and the largest PDE subfamily with over 35 different isoforms and as such, the most widely characterized PDE isoenzyme. It is the predominant isoenzyme in the majority of inflammatory cells, and the airways smooth muscle and is implicated in inflammatory airways disease. PDE4 inhibitor roflumilast has been approved for the treatment of chronic obstructive lung disease.
Hao N., et al., 2020, Mol Cancer Ther 19(3): 868-881.
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