Probing ProteinProtein Interactions with FRETFLIM

The quantification of molecular interactions or conformational changes can conveniently be studied by using F�rster Resonance Energy Transfer (FRET) as a spectroscopic ruler. The FRET phenomenon describes the transfer of energy from a donor to an acceptor molecule, if they are in close proximity (<10 nm). The most straightforward method to measure FRET is Fluorescence Lifetime Imaging Microscopy (FLIM). In this chapter, we will describe an application of FRET using FLIM to monitor the hexamer formation of CrFP/eYFP-labeled Arabidopsis thaliana cell division cycle protein (AtCDC48) expressed in plant protoplasts.