High Throughput Live-Cell FRET Binding Assay by Flow Cytometry

Abstract

Ratiometric live-cell fluorescence resonance energy transfer (FRET) assays hold significant promise for measuring binding affinity of protein-protein interactions. Compared to conventional in vitro binding assays, live-cell FRET gauges interaction within a native regime that favors proper folding, permits physiological modulation of association, and obviates costly purification of proteins. Previously, our lab developed a live-cell FRET binding assay (three-cube method) for cell-by-cell epifluorescence and confocal microscopy (Neuron31:973, J Microsc233:192). While accurate (Nat Commun4:1717), this approach is comparatively slow and labor intensive, challenging high-throughput application. Here we transformed this live-cell FRET approach into the realm of flow cytometry, allowing full binding curves encompassing data from thousands of cells within seconds. We were able to get similar results between the new configuration and microscope-based setup. As an example, we characterized well-studied interactions between Venus-tagged SH3 domains of Gad and a suite of Cerulean-tagged binding peptides. Three such interactions are illustrated with a range of affinities, in close agreement with in vitro iTC measurements. This new flow-cytometry approach (33-FRET-HTS) opens new possibilities for profiling protein-protein interactions in the native context.View Large Image | View Hi-Res Image | Download PowerPoint Slide