- Authors:
-
- Mathiesen, Jesper Mosolff ;
- Vedel, Line ;
-
Bräuner-Osborne, Hans
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0000-0001-9495-7388
Medicinal Chemistry Research, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
- DOI:
- 10.1016/B978-0-12-407865-9.00011-X
- Abstract:
- Cyclic adenosine monophosphate (cAMP) is a common second messenger that mediates numerous biological responses. Intracellular cAMP levels are increased by activation of G(s)-coupled G protein-coupled receptors (GPCRs) and decreased by activation of G(i)-coupled GPCRs via the adenylyl cyclase. Many end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled ß(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format.
- Type:
- Journal article
- Language:
- English
- Published in:
- Methods in Enzymology, 2013, Vol 522, p. 191-207
- Main Research Area:
- Medical science
- Publication Status:
- Published
- Review type:
- Undetermined
- Submission year:
- 2013
- Scientific Level:
- Scientific
- ID:
- 235795845
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