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Cytosolic expression of Green Fluorescent Protein (GFP) and its derivatives ,,, in the yeast Saccharomyces cerevisiae: Detection in vivo using ,,, the Varian Cary Eclipse

ic fluorescent protein (See the Clontech Web Site3 for a comprehensive list of excitation/emission maxima of fluorescent proteins.) Emission scans were recorded for GFP, BFP, CFP, YFP and DsRed. Operating parameters for scans of DsRed are given in Figure 2.

Results

Emission spectra for each of the fluorescent proteins are shown in Figure 3.

Discussion

Spectra from all fluorescent proteins expressed in the cytosol of the yeast cells were easily recorded and correspond to expected characteristics. Noise produced by cellular autofluorescence and scatter was negligible, mainly due to internal filters fitted to both excitation and emission monochromators on the Varian Cary Eclipse. Reducing autofluorescence (from either sample of interest or media) is of paramount importance when using fluorescent proteins that have either low fluorescent intensity, or are excited by high energy UV light (such as BFP). UV light initiates a strong fluorescent response from many components of the cellular environment and must be minimized to prevent masking of emission from target fluorescent proteins, especially when subtle protein interactions are being monitored7.

Conclusion

The Varian Cary Eclipse with multicell and temperature control accessories provides a simple, rapid and reproducible assembly with which to measure and characterise different fluorescent proteins expressed in whole cells. The opportunity now exists to specifically tag proteins of interest within cells using different GFPs and monitor interactions simultaneously using multiwavelength readings which allow automated excitation and
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