Journal Article

FZJ-2020-03033

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

Gruteser, N. ; Kohlhas, V. ; Balfanz, S.FZJ* ; Franzen, A.FZJ* ; Günther, A. ; Offenhäusser, A.FZJ* ; Müller, F.FZJ* ; Nikolaev, V. ; Lohse, M. J. ; Baumann, A. (Corresponding author)FZJ*

2020
BioMed Central London

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Please use a persistent id in citations: http://hdl.handle.net/2128/25559  doi:10.1186/s12896-020-00633-y

Abstract: 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.

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Contributing Institute(s):

1. Molekular- und Zellphysiologie (IBI-1)
2. Bioelektronik (IBI-3)

Research Program(s):

1. 552 - Engineering Cell Function (POF3-552) (POF3-552)

Appears in the scientific report 2020

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