%0 Journal Article
%A Gruteser, Nadine
%A Kohlhas, Viktoria
%A Balfanz, Sabine
%A Franzen, Arne
%A Günther, Anne
%A Offenhäusser, Andreas
%A Müller, Frank
%A Nikolaev, Viacheslav
%A Lohse, Martin J.
%A Baumann, Arnd
%T Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
%J BMC biotechnology
%V 20
%N 1
%@ 1472-6750
%C London
%I BioMed Central
%M FZJ-2020-03033
%P 47
%D 2020
%X BackgroundApproximately 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.ResultsTo 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.ConclusionsWe 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.
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:32854679
%U <Go to ISI:>//WOS:000567175700001
%R 10.1186/s12896-020-00633-y
%U https://juser.fz-juelich.de/record/878733