Recombinant Human Phosphodiesterase 4A/PDE4A Protein, CF Summary
Pro331-Met723, with an N-terminal Met and a C-terminal 6-His tag
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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 freeze-thaw cycles.
- 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
- 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.
- 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.
- Dilute rhPDE4A to 0.025 µg/mL in Assay Buffer.
- Create a substrate mixture containing 1 µg/mL Coupling Phosphatase 2 and 0.4 mM cAMP in Assay Buffer.
- Load 25 µL of each dilution of the standard curve into a plate. Include a Control containing 25 µL of Assay Buffer.
- Load 25 µL of the 0.025 µg/mL rhPDE4A into the plate.
- Add 25 µL of the substrate mixture to all wells, including the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 30 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells, including curve. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Phosphate released* (nmol) x (1000 pmol/nmol)|
|Incubation time (min) x amount of enzyme (µg)|
*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
Background: Phosphodiesterase 4A/PDE4A
Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentration of the second messengers, cAMP and cGMP (1-3). Phosphodiesterase 4, subtype A (PDE4A) is a rolipram-sensitive 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 anti-inflammatory therapeutics for treatment of asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, multiple sclerosis, type 2 diabetes, and potentially several CNS disorders (1-5). R&D Systems’ recombinant human PDE4A corresponds to the catalytic domain (6).
- Bender A.T. and J.A. Beavo (2006) Pharmacol. Rev. 58:488.
- Menniti F.S. et al. (2006) Nat. Rev. Drug Discov. 5:660.
- Omori K. and J. Kotera (2007) Circ. Res. 100:309.
- Spina D. (2008) Br. J. Pharmacol. 155:308.
- Wang, H. et al. (2007) Biochem. J. 408:193.
- Lario, P.I. et al. (2001) Arch. Biochem. Biophys. 394:54.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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Glycosyltransferase Activity Assays
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