TY  - JOUR
AU  - Gruteser, Nadine
AU  - Kohlhas, Viktoria
AU  - Balfanz, Sabine
AU  - Franzen, Arne
AU  - Günther, Anne
AU  - Offenhäusser, Andreas
AU  - Müller, Frank
AU  - Nikolaev, Viacheslav
AU  - Lohse, Martin J.
AU  - Baumann, Arnd
TI  - Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
JO  - BMC biotechnology
VL  - 20
IS  - 1
SN  - 1472-6750
CY  - London
PB  - BioMed Central
M1  - FZJ-2020-03033
SP  - 47
PY  - 2020
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:32854679
UR  - <Go to ISI:>//WOS:000567175700001
DO  - DOI:10.1186/s12896-020-00633-y
UR  - https://juser.fz-juelich.de/record/878733
ER  -