Recombinant Human
Phosphodiesterase 4A/PDE4A Catalog Number: 7767-PE
DESCRIPTION Source Spodoptera frugiperda, Sf 21 (baculovirus)derived Pro331Met723, with an Nterminal Met and a Cterminal 6His tag Accession # P27815
Nterminal Sequence Pro331 Analysis Predicted Molecular 46 kDa Mass
SPECIFICATIONS SDSPAGE 4448 kDa, reducing conditions
Activity Measured by its ability to convert cAMP to 5'AMP. The specific activity is >28,000 pmol/min/μg, as measured under the described conditions.
Endotoxin Level <0.01 EU per 1 μg of the protein by the LAL method.
Purity >95%, by SDSPAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane. Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. See Certificate of Analysis for details.
Activity Assay Protocol
Materials l Assay Buffer (1X): 20 mM Tris, 1 mM MgCl2, 1 mM DTT, 0.01538% CHAPS, pH 7.5 l Recombinant Human Phosphodiesterase 4A/PDE4A (rhPDE4A) (Catalog # 7767PE) l Adenosine 3’,5’cyclic monophosphate (cAMP) (Sigma, Catalog # A6885) 0.1 M stock in deionized water l Sialyltransferase Activity Kit (Catalog # EA002) l 96well Clear Plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Assay 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 onehalf serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.0392.5 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: Phosphate released* (nmol) x (1000 pmol/nmol) Specific Activity (pmol/min/µg) = Incubation time (min) x amount of enzyme (µg) *Derived from the phosphate standard curve using linear or 4parameter fitting and adjusted for Control.
Final Assay Per Reaction: Conditions l rhPDE4A: 0.000625 μg l Coupling Phosphatase 2: 25 ng l cAMP: 0.2 mM
PREPARATION AND STORAGE Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. Stability & Storage Use a manual defrost freezer and avoid repeated freezethaw cycles. l 6 months from date of receipt, 70 °C as supplied. l 3 months, 70 °C under sterile conditions after opening.
Rev. 2/6/2018 Page 1 of 2
Recombinant Human
Phosphodiesterase 4A/PDE4A Catalog Number: 7767-PE
BACKGROUND Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentration of the second messengers, cAMP and cGMP (13). Phosphodiesterase 4, subtype A (PDE4A) is a rolipramsensitive member of the cyclic phosphodiesterase family and catalyzes the specific hydrolysis of cAMP to 5'AMP. Numerous inhibitors of PDE4 have been studied and considered as antiinflammatory therapeutics for treatment of asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, multiple sclerosis, type 2 diabetes, and potentially several CNS disorders (15). R&D Systems’ recombinant human PDE4A corresponds to the catalytic domain (6).
References: 1. Bender A.T. and J.A. Beavo (2006) Pharmacol. Rev. 58:488. 2. Menniti F.S. et al. (2006) Nat. Rev. Drug Discov. 5:660. 3. Omori K. and J. Kotera (2007) Circ. Res. 100:309. 4. Spina D. (2008) Br. J. Pharmacol. 155:308. 5. Wang, H. et al. (2007) Biochem. J. 408:193. 6. Lario, P.I. et al. (2001) Arch. Biochem. Biophys. 394:54.
PRODUCT SPECIFIC NOTICES Coomassie is a registered trademark of Imperial Chemical Industries Ltd.
Rev. 2/6/2018 Page 2 of 2