Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
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Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides. / Gruteser, Nadine; Kohlhas, Viktoria; Balfanz, Sabine; Franzen, Arne; Günther, Anne; Offenhäusser, Andreas; Müller, Frank; Nikolaev, Viacheslav; Lohse, Martin J; Baumann, Arnd.
In: BMC BIOTECHNOL, Vol. 20, No. 1, 27.08.2020, p. 47.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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T1 - Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
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
PY - 2020/8/27
Y1 - 2020/8/27
N2 - 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.
AB - 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.
U2 - 10.1186/s12896-020-00633-y
DO - 10.1186/s12896-020-00633-y
M3 - SCORING: Journal article
C2 - 32854679
VL - 20
SP - 47
JO - BMC BIOTECHNOL
JF - BMC BIOTECHNOL
SN - 1472-6750
IS - 1
ER -