The present study aimed to detect and monitor changes in FRET between BFP and GFP in cytosolic lysates of yeast cells using the Varian Cary Eclipse.
Materials and Methods
(For part numbers see reference6)
Varian Cary Eclipse fluorescence spectrophotometer
Peltier-thermostatted multicell holder (with electromagnetic stirring)
Magnetic stirrer bars
YRD15 (MATa, his3, ura3, leu2, p+) of the yeast S. cerevisiae was the parental strain used in this study. A gene cassette was constructed encoding BFP and GFP linked by a 27 amino acid peptide linker that contains a recognition site for the protease trypsin. This cassette was cloned into the yeast expression plasmid pAS1N for cytosolic expression and transformed into the yeast strain YRD15 as previously described7. Transformants were plated out on yeast minimal medium (0.75% yeast minimal medium w/o amino acids, 2% glucose, 1.5% agar) with growth supplements as required and grown at 28C for 35 days.
Yeast cells were washed twice in 1ml MilliQ water before being lysed using
Y-PER (Progen) as per the manufacturers instructions. Lysates were preferred
over whole cells to allow protease digestion of the peptide linker. Y-PER lysates
(10l) were diluted with 1.2ml Tris/HCl pH 8 and placed in disposable fluorescence
cuvettes (Sarstedt) of the mult