1. Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.
- Author
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Gruteser N, Kohlhas V, Balfanz S, Franzen A, Günther A, Offenhäusser A, Müller F, Nikolaev V, Lohse MJ, and Baumann A
- Subjects
- Biosensing Techniques methods, Cell Culture Techniques, Cloning, Molecular, Cyclic AMP, Escherichia coli genetics, Guanine Nucleotide Exchange Factors, Hydrogen-Ion Concentration, Receptors, G-Protein-Coupled, Recombinant Proteins, Fluorescence, Fluorescence Resonance Energy Transfer methods, Nucleotides, Cyclic genetics
- 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.
- Published
- 2020
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