FAM-cAMP PDE IV substrate *Green Fluorescence*

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Chemical structure for FAM-cAMP PDE IV substrate *Green Fluorescence*
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Unit Size: Cat No: Price (USD): Qty:
0.5 umol 13602 $295


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)492/516
MW~800
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Enzyme Detection
Phosphodiesterases
Related cAMP GPCR Assays
Secondary Reagents
This green fluorescent cAMP derivative is a specific substrate for phosphodiesterase (PDE) IV. It can be used for assaying PDE IV activities or screening PDE IV inhibitors in combination with anti-cAMP antibody in a FRET readout or FP format. PDE is a group of enzymes that degrade the second messenger molecules: cyclic nucleotides cAMP and cGMP. They regulate the localization, duration, and amplitude of cyclic nucleotide signaling within subcellular domains. PDEs are therefore important regulators of signal transduction mediated by these second messenger molecules. PDE enzymes are often targets for pharmacological inhibition due to their unique tissue distribution, structural and functional properties. Inhibitors of PDE can prolong or enhance the effects of physiological processes mediated by cAMP or cGMP by inhibition of their degradation by PDE. PDE inhibitors have been identified as new potential therapeutics in areas such as pulmonary arterial hypertension, coronary heart disease, dementia, depression and schizophrenia.




Calculators
Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of FAM-cAMP PDE IV substrate *Green Fluorescence* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.



Molarity calculator

Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =
 






Spectrum Advanced Spectrum Viewer

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Wavelength (nm)





Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Important notes
Following protocol only provides a guideline, and should be modified according to your specific needs.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:490 nm
Emission:525 nm
Cutoff:515 nm
Recommended plate:Solid black
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. FAM-cAMP PDE IV stock solution (1 mM):
Make a 1 mM stock solution by adding 500 µL of DMSO into the vial of 0.5 umol FAM-cAMP PDE IV substrate.

Preparation of working solution

FAM-Cyclic-3’, 5’-AMP PDE IV substrate assay solution (2X):
Make 2X FAM-Cyclic-3’, 5’-AMP PDE IV substrate assay solution by diluting 1 mM FAM-Cyclic-3’ ,5’-AMP PDE IV substrate stock solution into your PDE buffer (such as 10 mM Tris-HCl, pH 7.4, 10 mM Mg Cl2, 1 mM MnCl2) to make a 200 - 400 nM solution. Note: Make only sufficient quantity needed for the assay.

Sample experimental protocol
  1. Mix equal volume of the PDE IV standards or samples with 2X FAM-Cyclic-3’ ,5’-AMP PDE IV substrate assay solution, and incubate at room temperature for at least 1 hour.

  2. Monitor the fluorescence polarization at Ex/Em = 490/525 nm.
Example data analysis and figures

Figure 1. Chemical structure for FAM-cAMP PDE IV substrate *Green Fluorescence*
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

cAMP is a ligand for the tandem GAF domain of human phosphodiesterase 10 and cGMP for the tandem GAF domain of phosphodiesterase 11
Authors: Gross-Langenhoff M, Hofbauer K, Weber J, Schultz A, Schultz JE.
Journal: J Biol Chem (2006): 2841

cAMP phosphodiesterase activity evaluation in human carcinoma of salivary glands
Authors: Spoto G, della Malva M, Rubini C, Fioroni M, Piattelli A, Serra E, Di Nicola M, Santoleri F.
Journal: Nucleosides Nucleotides Nucleic Acids (2006): 1113

cAMP phosphodiesterase-4A1 (PDE4A1) has provided the paradigm for the intracellular targeting of phosphodiesterases, a process that underpins compartmentalized cAMP signalling
Authors: Huston E, Houslay TM, Baillie GS, Houslay MD.
Journal: Biochem Soc Trans (2006): 504

Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi
Authors: Diaz-Benjumea R, Laxman S, Hinds TR, Beavo JA, Rascon A.
Journal: Biochem J (2006): 305

Compartmentalization of cAMP-Dependent Signaling by Phosphodiesterase-4D Is Involved in the Regulation of Vasopressin-Mediated Water Reabsorption in Renal Principal Cells
Authors: Stefan E, Wiesner B, Baillie GS, Mollajew R, Henn V, Lorenz D, Furkert J, Santamaria K, Nedvetsky P, Hundsrucker C, Beyermann M, Krause E, Pohl P, Gall I, Macintyre AN, Bachmann S, Houslay MD, Rosenthal W, Klussmann E.
Journal: J Am Soc Nephrol. (2006)

DdPDE4, a novel cAMP-specific phosphodiesterase at the surface of dictyostelium cells
Authors: Bader S, Kortholt A, Snippe H, Van Haastert PJ.
Journal: J Biol Chem (2006): 20018

Expression and Activity of cAMP Phosphodiesterase Isoforms in Pulmonary Artery Smooth Muscle Cells from Patients with Pulmonary Hypertension: Role for PDE1
Authors: Murray F, Patel HH, Suda RY, Zhang S, Thistlethwaite P, Yuan JX, Insel PA.
Journal: Am J Physiol Lung Cell Mol Physiol. (2006)

Expression of cAMP and cGMP-phosphodiesterase isoenzymes 3, 4, and 5 in the human clitoris: immunohistochemical and molecular biology study
Authors: Oelke M, Hedlund P, Albrecht K, Ellinghaus P, Stief CG, Jonas U, Andersson KE, Uckert S.
Journal: Urology (2006): 1111

Expression of cAMP and cGMP-phosphodiesterase isoenzymes 3, 4, and 5 in the human clitoris: immunohistochemical and molecular biology study
Authors: Sampaio F.
Journal: Int Braz J Urol (2006): 483

Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+
Authors: Huston E, Gall I, Houslay TM, Houslay MD.
Journal: J Cell Sci (2006): 3799


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