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@ARTICLE{Gruteser:878733,
      author       = {Gruteser, Nadine and Kohlhas, Viktoria and Balfanz, Sabine
                      and Franzen, Arne and Günther, Anne and Offenhäusser,
                      Andreas and Müller, Frank and Nikolaev, Viacheslav and
                      Lohse, Martin J. and Baumann, Arnd},
      title        = {{E}stablishing a sensitive fluorescence-based
                      quantification method for cyclic nucleotides},
      journal      = {BMC biotechnology},
      volume       = {20},
      number       = {1},
      issn         = {1472-6750},
      address      = {London},
      publisher    = {BioMed Central},
      reportid     = {FZJ-2020-03033},
      pages        = {47},
      year         = {2020},
      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.},
      cin          = {IBI-1 / IBI-3},
      ddc          = {610},
      cid          = {I:(DE-Juel1)IBI-1-20200312 / I:(DE-Juel1)IBI-3-20200312},
      pnm          = {552 - Engineering Cell Function (POF3-552)},
      pid          = {G:(DE-HGF)POF3-552},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:32854679},
      UT           = {WOS:000567175700001},
      doi          = {10.1186/s12896-020-00633-y},
      url          = {https://juser.fz-juelich.de/record/878733},
}