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| Item Type: | Article |
|---|---|
| Title: | Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides |
| Creators Name: | Gruteser, N., Kohlhas, V., Balfanz, S., Franzen, A., Günther, A., Offenhäusser, A., Müller, F., Nikolaev, V., Lohse, M.J. and Baumann, A. |
| 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 |
| Source: | BMC Biotechnology |
| ISSN: | 1472-6750 |
| Publisher: | BioMed Central |
| Volume: | 20 |
| Number: | 1 |
| Page Range: | 47 |
| Date: | 27 August 2020 |
| Official Publication: | https://doi.org/10.1186/s12896-020-00633-y |
| PubMed: | View item in PubMed |
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