Macromolecular Crowding Measurements with Genetically Encoded Probes Based on Förster Resonance Energy Transfer in Living Cells

Methods Mol Biol. 2020;2175:169-180. doi: 10.1007/978-1-0716-0763-3_12.


Genetically encoded Förster resonance energy transfer (FRET)-based probes allow a sensitive readout for different or specific parameters in the living cell. We previously demonstrated how FRET-based probes could quantify macromolecular crowding with high spatio-temporal resolution and under various conditions. Here, we present a protocol developed for the use of FRET-based crowding probes in baker's yeast, but the general considerations also apply to other species, as well as other FRET-based sensors. This method allows straightforward detection of macromolecular crowding under challenging conditions often presented by living cells.

Keywords: Förster resonance energy transfer; Genetically encoded probes; Living cells; Macromolecular crowding; Sensors.