Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET

James M Pemberton; Qian Liu; David W Andrews

doi:10.21769/BioProtoc.3401

Measuring Small-molecule Inhibition of Protein Interactions in Live Cells Using FLIM-FRET

Bio Protoc. 2019 Oct 20;9(20):e3401. doi: 10.21769/BioProtoc.3401.

Authors

James M Pemberton^{1

2}, Qian Liu¹, David W Andrews^{1

2}

Affiliations

¹ Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada, M4N 3M5.
² Department of Medical Biophysics, University of Toronto, Ontario, Canada.

Abstract

This protocol was designed to quantitatively measure small-molecule displacement of proteins in live mammalian cells using fluorescence lifetime imaging microscopy-Förster resonance energy transfer (FLIM-FRET). Tumour cell survival is often dependent on anti-apoptotic proteins, which bind to and inhibit pro-apoptotic proteins, thus preventing apoptosis. Small-molecule inhibitors that selectively target these proteins (termed BH3-mimetics) are therefore a promising avenue for the treatment of several cancers. Previous techniques used to study the efficacy of these drugs often use truncated versions of both pro- and anti-apoptotic proteins, as they are membrane bound and hydrophobic in nature. As a result, the true efficacy of these drugs to displace full-length pro-apoptotic proteins in their native environment within a cell is poorly understood. This protocol describes FLIM-FRET methods to directly measure the displacement (or lack of displacement) of full-length Bcl-2 family proteins in live mammalian cells.

Keywords: Apoptosis; BH3-mimetic; Bad; Bcl-2; Bcl-XL; Displacement; FLIM; FRET; Live cell; Resistance.