Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
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METHODOLOGY ARTICLE
Open Access
Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides Nadine Gruteser1, Viktoria Kohlhas1,2, Sabine Balfanz1, Arne Franzen1, Anne Günther1,3, Andreas Offenhäusser4, Frank Müller1, Viacheslav Nikolaev5, Martin J. Lohse6,7 and Arnd Baumann1*
Abstract Background: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. Results: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. Conclusions: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses. Keywords: Cyclic nucleotide quantification, Cell-based assay, Epac1-camps, Optogenetic sensor, Signaling
Background Signaling within and between cells often invokes transient and spatially restricted changes of intracellular second messenger concentrations. In addition to calcium (Ca2+) signals, changes in intracellular concentrations of cyclic adenosine 3′,5′-monophosphate (cAMP) or cyclic guanosine 3′,5′-monophosphate (cGMP) play an important role in cell signaling. As second messengers, cyclic nucleotides are known to control the activity of * Correspondence: [email protected] 1 Institute of Biological Information Processing (Molecular and Cellular Physiology, IBI-1), Forschungszentrum Jülich, 52428 Jülich, Germany Full list of author information is available at the end of the article
protein kinases and phosphatases as well as Popeye domain containing (Popdc [6];) proteins. Kinases and phosphatases then modulate the phosphorylation status of downstream proteins participating, e.g., in cellular trafficking, metabolic uptake mechanisms as well as regulation of gene transcription (reviewed in: [22, 37]). Furthermore, cyclic nucleotides have been shown to either directly activate cyclic nucleotide-gated ion channels in many cells, including photoreceptors and sensory neurons (revie
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