[Yeast] Efflux of fluorophores in Yeast

da49 At drexel.edu via yeast%40net.bio.net (by da49 At drexel.edu)
Mon Nov 27 13:47:34 EST 2006


Hi,

Our lab is involved in studying efflux of fluorescent compounds in yeast (the strain we are using is S. cerevisiae YALF-A1 and its isogenic segregant YALF-I1 (Wolfger, H. et. al. FEBS Lett, (1997) 418: 269-274). The cells are grown in YPD media and then resuspended in PBS buffer (prior to the addition of fluorophores – media such as YPD were giving an extremely high background during fluorimetric reading).We are trying to do a time-based study, i.e. look at uptake/efflux over time in a fluorimeter with a slit width of 2-4 nm. The graphs that we get are plotted in terms of Counts/sec (y-axis) vs. Time x-axis).

I was having some trouble with repeatability of the experiments. While trying to run repeats (same day and conditions), I am unable to get overlapping curves. Normalizing the curve (0% for PBS buffer only and 100% for cells with fluorophore solubalized with Triton-X100) did not help. We have tried controlling/lowering the temperature and working with spheroplasts (instead of intact cells) as well, but it did not make any difference. We also tried looking at wavelength shift during uptake (Gaskova, et. al. Int. J. Biochem. Cell Biol. (2002) 34: 931-937), but did not get satisfactory results with this technique either.

I was hoping that someone with experience with yeast efflux/ fluorescence could help me out with this problem. I was wondering if the “spread” in fluorimetric readings that we were seeing is because of the organism being studied or is commonly seen in fluorimetric assays.

I would greatly appreciate any help in this matter.

Thanking you,
Yours truly,

Deep Agnani
Ph.D. Student.
Department of Bioscience and Biotechnology
Drexel University
Philadelphia, PA, USA





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