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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

RET could be accurately monitored in cytosolic lysates from yeast cells. Cleavage of the BFP-GFP fusion using trypsin (Peltier temperature controlled at 25C) demonstrated that GFP emission was due to FRET and not to direct stimulation of GFP by the excitation wavelength. This is demonstrated by the scans shown in Figure 3 that depict the green (FRET) peak becoming smaller (as BFP is cleaved from GFP) with successive scans over a period of 33 minutes.


Conclusion

Conclusion The Varian Cary Eclipse with Peltier temperature control and multicell accessories provide a simple and accurate assembly with which to monitor cell function at the protein level in cytosolic lysates from yeast cells. The opportunity now exists to use this model as a platform with which to investigate protein-protein interactions in response to external or internal stimuli in living cells.


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