Measuring small-molecule inhibition of protein interactions in live cells using flim-fret

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 FLIMFRET methods to directly measure the displacement (or lack of displacement) of full-length Bcl-2 family proteins in live mammalian cells.