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TAMRA-cAMP PDE IV substrate *Red Fluorescence*
This red 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.
Example protocol
AT A GLANCE
Important notes
Following protocol only provides a guideline, and should be modified according to your specific needs.
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. TAMRA-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 TAMRA-cAMP PDE IV substrate. Note: Unused stock solution can be stored at -20 oC in dark place in single aliquotes.
PREPARATION OF WORKING SOLUTION
TAMRA-Cyclic-3’, 5’-AMP PDE IV substrate assay solution (2X):
Make 2X TAMRA-Cyclic-3’, 5’-AMP PDE IV substrate assay solution by diluting 1 mM TAMRA-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
- Mix equal volume of the PDE IV standards or samples with 2X TAMRA-Cyclic-3’ ,5’-AMP PDE IV substrate assay solution, and incubate at room temperature for at least 1 hour.
- Monitor the fluorescence polarization at Ex/Em = 540/590 nm.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (260 nm) | Correction Factor (280 nm) |
| FAM-cAMP PDE IV substrate *Green Fluorescence* | 493 | 517 | 83000 | 0.32 | 0.178 |
References
View all 34 references: Citation Explorer
Importance of cAMP-response element-binding protein in regulation of expression of the murine cyclic nucleotide phosphodiesterase 3B (Pde3b) gene in differentiating 3T3-L1 preadipocytes
Authors: Liu H, Tang JR, Choi YH, Napolitano M, Hockman S, Taira M, Degerman E, Manganiello VC.
Journal: J Biol Chem (2006): 21096
Authors: Liu H, Tang JR, Choi YH, Napolitano M, Hockman S, Taira M, Degerman E, Manganiello VC.
Journal: J Biol Chem (2006): 21096
Immunohistochemical distribution of cAMP- and cGMP-Phosphodiesterase (PDE) isoenzymes in the human prostate
Authors: Sampaio FJ., undefined
Journal: Int Braz J Urol (2006): 368
Authors: Sampaio FJ., undefined
Journal: Int Braz J Urol (2006): 368
Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5
Authors: Bolger GB, Baillie GS, Li X, Lynch MJ, Herzyk P, Mohamed A, Mitchell LH, McCahill A, Hundsrucker C, Klussmann E, Adams DR, Houslay MD.
Journal: Biochem J (2006): 23
Authors: Bolger GB, Baillie GS, Li X, Lynch MJ, Herzyk P, Mohamed A, Mitchell LH, McCahill A, Hundsrucker C, Klussmann E, Adams DR, Houslay MD.
Journal: Biochem J (2006): 23
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
Authors: Huston E, Houslay TM, Baillie GS, Houslay MD.
Journal: Biochem Soc Trans (2006): 504
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)
Authors: Murray F, Patel HH, Suda RY, Zhang S, Thistlethwaite P, Yuan JX, Insel PA.
Journal: Am J Physiol Lung Cell Mol Physiol. (2006)
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