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Monitoring fluorescence resonance energy transfer (FRET) between GFP,,, fusions in lysates of the yeast Saccharomyces cerevisiae using the ,,, Varian,,, Cary Eclipse

icell holder positioned within the sample chamber of the Varian Cary Eclipse. The temperature within the cuvettes was set to 25C to promote cleavage of the peptide linker by trypsin (Figure 2). Using the Scan application, BFP was specifically excited using light of 360nm, and emission spectra for the fusion protein were recorded from the range 400550nm. Further emission scans were recorded over time after the addition of 0.25g trypsin.

Results

Emission spectra of the BFP-GFP fusion protein following 360nm excitation are shown in Figure 3. An initial spectrum was taken at time = 0 min, then trypsin was added and spectra were recorded at the times indicated. GFP emission (~510nm) is seen upon specific excitation of BFP alone (360nm), indicative of FRET. Spectral characteristics of FRET (indicated by the green peak at 510nm) progressively disappeared following the addition of trypsin, which cleaves the peptide linker that tethers the GFPs. A small increase is seen in BFP emission as FRET diminishes.

Discussion

An ideal strategy to monitor the interactions of proteins in living systems involves detection of FRET between BFP and GFP bound to target species of interest. For optimum selectivity and sensitivity of detection of fluorescence in applications such as this it is necessary to minimise (a) detection of cellular autofluorescence and (b) photobleaching. These issues are addressed by internal filters (on both excitation and emission monochromators) and the Cary Eclipse xenon flash lamp respectively. The issue of photobleaching is extremely important and is discussed in a separate application note.

The data indicate (Figure 3) that F
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