FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP
Published in FEBS Letters, 2001
Recommended citation: Romero et al.", 2017 http://fraroco.github.io/files/FRomero_FEBSLETTERS_2017.pdf
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 EPAC1 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 CNBDs.
Recommended citation: Romero et al.”, 2017