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A Dohlman Lab Protocol

REFERENCE: Hoffman, G., Garrison, T. R., and Dohlman, H. G., Analysis of RGS proteins in Saccharomyces cerevisiae, Methods Enzymol. 344:617-631, 2002.

1. Grow a starter culture at 30 C shaking (250 rpm) until it reaches saturation.

  • The cells should be transformed with a yeast expression vector containing the lacZ gene under the control of the FUS1 promoter. Alternately, a strain can be used with the FUS1-lacZ reporter integrated into the genome. These materials are available from a number of yeast labs, including our own.

2. Using the saturated starter culture, inoculate 5 to 25 ml of the appropriate media.

  • Rarely, a transformed colony will not respond at all to pheromone. The cause is not known. To eliminate these “flatliner” strains pick several different colonies. Two hours after re-inoculation take an aliquot of each and perform a small scale assay (plus and minus 1 dose of alpha-factor). Discard any that fail to change color after substrate addition.

3. Grow at 30 C shaking (250 rpm) until the OD600 nm ~ 0.8 (this is usually done overnight).

  • This is the trickiest part of the assay, since it is difficult to get different strains to reach OD600 nm ~ 0.8 at the same time. The best way to handle this is to start a 2 ml starter culture about 3-4 days before the assay. The night before the assay, start a 10 to 25 ml intermediate culture. The morning of the assay, measure the absorbance of all of the intermediate cultures and dilute them down to an OD600 nm of 0.2 in pre-warmed media. The strains will now only have to go through two doublings and the amount of variance between them should be reduced. If the strains still reach OD600 nm ~ 0.8 at different times, it is acceptable to put strains which have reached OD600 nm ~ 0.8 on ice while waiting for the others.
  • Re-check the absorbance of all cultures before proceeding.

4. Aliquot 10 ul of alpha-factor at the appropriate concentrations into 96 well plates.

  • Each strain should be tested with 8 to 10 different concentrations of alpha-factor in triplicate.
  • A good set of final alpha-factor concentrations for testing an sst2delta mutant strain is: 0 , 0.00003 uM, 0.0001 uM… 0.3 uM. For strains expressing a mammalian or yeast RGS protein, the final concentrations should be 100-300 fold higher. The concentration of alpha-factor added to each well must be 10-fold higher than the desired final concentration.
  • Keep a frozen stock of alpha-factor and make a new set of dilutions each time the assay is performed, since alpha-factor is subject to degradation upon repeated freeze-thaw cycles or exposure to room temperature.
  • A multi-channel pipettor and a plastic pipettor basin (Fisher Scientific, #13681100) is helpful for aliquoting solutions.

5. Add 90 ul of cells to each well.

6. Incubate 90 min at 30 C, shaking gently.

7. During the incubation, prepare the FDG solution.

Solution #1: 1 mM FDG stock diluted in 25 mM PIPES (pH 7.2)

Solution #2: 0.5% Triton X-100 diluted in 250 mM PIPES (pH 7.2)

  • Mix Solution #1 and Solution #2 in equal amounts just prior to use, and pour into a clean pipettor basin.

FDG Stock: 10 mM FDG (Fluorescein di-b-D-galactopyranoside, Molecular Probes, #F-1179) in dimethyl sulfoxide (DMSO) (FDG should be stored in DMSO at -20 C; it is more stable in solution.)

8. After the 90 min incubation, add 20 ul FDG solution per well. Shake plates gently and briefly.

9. Cover in aluminum foil and incubate at 37 C until a bright yellow color appears in some of the wells.

  • This can take from 10 to 90 min. Do not incubate longer than 90 min.

10. Stop the reaction by adding 20 ul of 1 M Na2CO3 per well. Shake the plates gently and briefly.

11. Read the plates with a fluorescence multi-well plate reader using an excitation of 485 nm and an emission of 530 nm.

12. Read the absorbance and normalize for cell density.

  • Alternately, use the final absorbance values [obtained immediately before aliquoting cells into the 96 well plate (step 3)] to normalize for cell density.

Updated 01/23/02