Helmholtz Gemeinschaft


Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

PDF (Original Article) - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader

Item Type:Article
Title:Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
Creators Name:Gruteser, N. and Kohlhas, V. and Balfanz, S. and Franzen, A. and Günther, A. and Offenhäusser, A. and Müller, F. and Nikolaev, V. and 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
Publisher:BioMed Central
Page Range:47
Date:27 August 2020
Official Publication:https://doi.org/10.1186/s12896-020-00633-y
PubMed:View item in PubMed

Repository Staff Only: item control page


Downloads per month over past year

Open Access
MDC Library