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| Hauptseite > Publikationsdatenbank > Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides > print |
| Hauptseite > Publikationsdatenbank > Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides > print |
| 001 | 878733 | ||
| 005 | 20220930130249.0 | ||
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| 100 | 1 | _ | |a Gruteser, Nadine |0 P:(DE-Juel1)145762 |b 0 |
| 245 | _ | _ | |a Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides |
| 260 | _ | _ | |a London |c 2020 |b BioMed Central |
| 336 | 7 | _ | |a article |2 DRIVER |
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| 520 | _ | _ | |a 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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| 700 | 1 | _ | |a Günther, Anne |0 P:(DE-HGF)0 |b 4 |
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| 700 | 1 | _ | |a Lohse, Martin J. |0 P:(DE-HGF)0 |b 8 |
| 700 | 1 | _ | |a Baumann, Arnd |0 P:(DE-Juel1)131911 |b 9 |e Corresponding author |
| 773 | _ | _ | |a 10.1186/s12896-020-00633-y |g Vol. 20, no. 1, p. 47 |0 PERI:(DE-600)2052746-9 |n 1 |p 47 |t BMC biotechnology |v 20 |y 2020 |x 1472-6750 |
| 856 | 4 | _ | |u https://juser.fz-juelich.de/record/878733/files/SN-2020-00398-b.pdf |
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