FRET (fluorescence resonance energy transfer) is a physical process in which energy is transferred from one fluorophore (donor) to another (acceptor) by dipole-dipole interaction. A requisite for this process is a significant overlap of the donor emission spectrum and the acceptor absorption spectrum and the spatial proximity and orientation of two fluorophores (mostly within 10nm) (Figure 1). Applications utilizing FRET include the measure ment of receptor interactions, transcription factors and binding proteins or caspase- and kinase- activities used for biochemical or immunological processes. Further, the excitation/emission properties of tandem conjugate fluorophore pairs used in flow cytometry (e.g., PE-Cy5, PE-Cy7 or APC-Cy7) are based on FRET.
This protocol describes how the interaction of the proteins interferon regulatory factor 1 (IRF-1) and myeloid differentiation factor 88 (MyD88) can be measured and visualized using the fluorescent protein moieties cyan fluorescent protein (CFP) as donor and yellow fluorescent protein (YFP) as acceptor.