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 .