Robust evaluation of intermolecular FRET using a large Stokes shift fluorophore as a donor

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However," most of the popular FRET pairs suffer cross-excitation of the acceptor"," which could lead to false positives. To overcome this problem"," we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example"," we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely"," a change in the fl uorescence spectral shape implies evidence of FRET without other control experiments.

Download paper here

Recommended citation: Santana-Calvo et al.”, 2018