Universität zu Köln

Gruteser, Nadine, Kohlhas, Viktoria, Balfanz, Sabine, Franzen, Arne ORCID: 0000-0002-8731-674X, Guenther, Anne, Offenhaeusser, Andreas, Mueller, Frank, Nikolaev, Viacheslav, Lohse, Martin J. and Baumann, Arnd ORCID: 0000-0001-9456-7275 (2020). Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides. BMC Biotechnol., 20 (1). LONDON: BMC. ISSN 1472-6750

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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 Forster 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Gruteser, Nadine UNSPECIFIED UNSPECIFIED UNSPECIFIED Kohlhas, Viktoria UNSPECIFIED UNSPECIFIED UNSPECIFIED Balfanz, Sabine UNSPECIFIED UNSPECIFIED UNSPECIFIED Franzen, Arne UNSPECIFIED orcid.org/0000-0002-8731-674X UNSPECIFIED Guenther, Anne UNSPECIFIED UNSPECIFIED UNSPECIFIED Offenhaeusser, Andreas UNSPECIFIED UNSPECIFIED UNSPECIFIED Mueller, Frank UNSPECIFIED UNSPECIFIED UNSPECIFIED Nikolaev, Viacheslav UNSPECIFIED UNSPECIFIED UNSPECIFIED Lohse, Martin J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Baumann, Arnd UNSPECIFIED orcid.org/0000-0001-9456-7275 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-322505
DOI:	10.1186/s12896-020-00633-y
Journal or Publication Title:	BMC Biotechnol.
Volume:	20
Number:	1
Date:	2020
Publisher:	BMC
Place of Publication:	LONDON
ISSN:	1472-6750
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CAMP; ASSAY; FAMILY; AMP; RADIOIMMUNOASSAY; OCTOPAMINE; CHANNELS; CALCIUM; SIGNALS; GMP Multiple languages Biotechnology & Applied Microbiology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/32250