Recombinant Human Phosphodiesterase 4A/PDE4A Protein, CF

• Assay Buffer (1X): 20 mM Tris, 1 mM MgCl₂, 1 mM DTT, 0.01538% CHAPS, pH 7.5
• Recombinant Human Phosphodiesterase 4A/PDE4A (rhPDE4A) (Catalog # 7767-PE)
• Adenosine 3’,5’-cyclic monophosphate (cAMP) (Sigma, Catalog # A6885) 0.1 M stock in deionized water
• Sialyltransferase Activity Kit (Catalog # EA002)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Sialyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
2. Continue standard curve by performing six additional one‑half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039‑2.5 nmol per well due to volume loaded in step 5.
3. Dilute rhPDE4A to 0.025 µg/mL in Assay Buffer.
4. Create a substrate mixture containing 1 µg/mL Coupling Phosphatase 2 and 0.4 mM cAMP in Assay Buffer.
5. Load 25 µL of each dilution of the standard curve into a plate. Include a Control containing 25 µL of Assay Buffer.
6. Load 25 µL of the 0.025 µg/mL rhPDE4A into the plate.
7. Add 25 µL of the substrate mixture to all wells, including the standard curve.
8. Cover the plate with a plate sealer and incubate at 37 °C for 30 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of deionized water to all wells, including curve. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

• rhPDE4A: 0.000625 μg
• Coupling Phosphatase 2: 25 ng
• cAMP: 0.2 mM