Flow cytometric analysis of fluorescence resonance energy transfer: a tool for high-throughput screening of molecular interactions in living cells

Abstract

The study of cellular processes has been facilitated by the use of methods to detect molecular associations both in vivo and in vitro. An invaluable tool to study molecular associations associated with dynamic processes in living cells utilizes the phenomenon of fluorescence resonance energy transfer (FRET), together with selected fluorophores that are attached to molecules of interest. Many reports have utilized fluorophores conjugated to antibodies for FRET pairs. However, these methods are restricted to extracellular molecules and dependent upon the availability of appropriate antibodies. The recent development of green fluorescent protein (GFP) variants suitable for FRET has expanded the utility of this methodology by permitting the study of intracellular as well as extracellular processes. Combining FRET with flow cytometric analysis results in a powerful high-throughput assay for molecular associations. This article details the use of green fluorescent protein (GFP) mutants cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure the association of the signaling component TRAF2 with the TNFR-2 receptor to illustrate the versatility of this methodology.