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FRET‐based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide‐binding domain tagged with a CFP
Author(s) -
Romero Francisco,
SantanaCalvo Carmen,
SánchezGuevara Yoloxochitl,
Nishigaki Takuya
Publication year - 2017
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.12760
Subject(s) - förster resonance energy transfer , chemistry , fluorescence , nucleotide , biophysics , cyclic nucleotide binding domain , biochemistry , biology , physics , quantum mechanics , gene
The cyclic nucleotide‐binding domain ( CNBD ) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer ( FRET ) between an adenosine‐3′, 5′‐cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC 1 tagged with a cyan fluorescence protein ( CFP ). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBD s.