On using mCherry as a label for use in wide-field homo-FRET induced fluorescence anisotropy microscopy

Abstract

Förster Resonance Energy Transfer (FRET) can be used to determine the interactions between proteins in cells. When using identical donor and acceptor molecules, it is referred to as homo-FRET. In this thesis we explore the possibilities of homo-FRET induced fluorescence anisotropy microscopy, and in particular we compare mCherry and GFP as labeling proteins. We found that the effect of cell autofluorescence on the accuracy of our measurements was greatly reduced by measuring in the red part of the spectrum (using mCherry). We expect to be able to measure the anisotropy of mCherry in cells with an accuracy of about 0.002, although more experiments are necessary to confirm this. Furthermore this thesis serves as a simple guide to homo-FRET and fluorescence anisotropy microscopy, and includes an extensive measurement and data analysis protocol.