Fluorescence Recovery After Photo-bleaching (FRAP) and Fluorescence Loss In Photo-bleaching (FLIP) experiments to study protein dynamics during budding yeast cell division

Abstract

The easiness of tagging any protein of interest with a fluorescent marker together with the advance of fluorescence microscopy techniques enable researchers to study in great detail the dynamic behavior of proteins both in time and space in living cells. Two commonly used techniques are FRAP (Fluorescent Recovery After Photo-bleaching) and FLIP (Fluorescence Loss In Photo-bleaching). Upon single bleaching (FRAP) or constant bleaching (FLIP) of the fluorescent signal in a specific area of the cell, the intensity of the fluorophore is monitored over time in the bleached area and in surrounding regions; information is then derived about the diffusion speed of the tagged molecule, the amount of mobile versus immobile molecules as well as the kinetics with which they exchange between different parts of the cell. Thereby, FRAP and FLIP are very informative about the kinetics with which the different organelles of the cell separate into mother- and daughter-specific compartments during cell division. Here, we describe protocols for both FRAP and FLIP and explain how they can be used to study protein dynamics during cell division in the budding yeast Saccharomyces cerevisiae. These techniques are easily adaptable to other model organisms.