Please find the following protocol:

Yeast Cell Cycle Experiment: Time Course for Western Blots


Overview:

This protocol describes an experimental technique for measuring protein levels in yeast of throughout the cell cycle. Mata yeast cells are first arrested in G1 with alpha factor. After release from alpha factor, the cells are allowed to progress through the cell cycle. Aliquots are collected at various time points and processed for Western blotting.


Procedure:

1. Grow up 2 to 3 ml of saturated cultures of the yeast strains of interest in YPD Medium. These cultures can be kept on your bench top for several weeks and provide a source for inoculating new cultures.

2. Inoculate 25 ml of YPD Medium with the saturated yeast cultures and grow to log phase; that is to an optical density (OD) of 0.4 to 0.6 at 600 nm. For wild type cells, try inoculating with 35 μl of saturated yeast culture and shake overnight at room temperature. Two cultures can be started the night before, using different amounts of saturated culture to ensure that one of them will be at the desired Optical Density (OD). Another option is to inoculate about 50 ml with 40 μl, and then pellet the cells in the morning and resuspend them in the appropriate amount of YPD to give the desired OD.

3. Add alpha factor to arrest the cells. For BAR+ cells add alpha factor to 20 μg/ml. For bar- cells add alpha factor to 1 μg/ml. The Bar1 protein is a protease secreted into the periplasmic space of Mata cells that cleaves and inactivates α factor. Mata bar1- cells are supersensitive to α factor-induced G1 arrest.

4. Incubate at room temperature for 3 to 3.5 hours with shaking. Ninety to 99% of the cells should be arrested with schmoos. bar- cells can be incubated for even longer without breaking through the arrest. BAR+ cells will start to break through the arrest after 3.5 hours, so don't let them go too long.

5. Take 1.6 ml of culture and pellet the cells in a screw-top microfuge tube by centrifuging for 1 min at maximum speed in a microcentrifuge.

6. Remove the supernatant, screw on a cap, and freeze in Liquid Nitrogen. This is the zero time point.

7. Wash away the alpha factor in the remaining culture by centrifuging the cells for 3 min at 3,500 rpm in the clinical centrifuge in 15 or 50 ml conical tubes.

8. Resuspend cells in 15 to 20 ml YPD Medium.

9. Centrifuge the cells for 3 min at 3,500 rpm in the clinical centrifuge in 15 or 50 ml conical tubes. Ecm mutants give a very flocculent pellet, which will go down the drain if you pour off the supernatant. For these mutants, it is best to aspirate the supernatant. You should be able to complete the washes within 9 min.

10. Resuspend the pellets to the original volume in YPD and transfer to a small culture flask.

11. Incubate at 30°C.

12. Take 1.6 ml samples every 10 min and process the same way as the zero time point.

Once all of the samples have been collected they can be stored at -80°C.

13. Take the cells out of -80°C, remove the caps, and add 1 cap full of acid-washed glass beads.

14. Add 150 μl of Sample Buffer with PMSF containing 1X LPC.

15. Vortex samples vigorously for 2 min. Do these steps as quickly as possible and in batches of 8 tubes. Try not to let the cell pellets thaw before they have been lysed by vortexing. Place the tubes on ice.

16. Incubate cell lysates in a boiling water bath for 3 to 5 min.

17. Centrifuge 5 min at maximum speed in microcentrifuge.

18. Load 10-15 μl sample per lane on a polyacrylamide gel.

19. Continue with Western Blotting.



Solutions:

Sample Buffer with PMSF
70 mM Tris
1% (w/v) 2-Mercaptoethanol or 1.5% (w/v) DTT
2% (w/v) SDS
0.1% (w/v) Bromophenol Blue
10% (v/v) Glycerol
2 mM Phenylmethylsulfonyl Fluoride (PMSF)
(CAUTION Biohazard!)

YPD Medium
Autoclave 20 min, cool to room temperature
10 g/liter Yeast Extract
20 g/liter Peptone
20g/liter Glucose (Dextrose)

LPC Stock (1000X)
Make up in DMSO (CAUTION Biohazard!)
10 mg/ml Chymostatin
10 mg/ml Pepstatin
10 mg/ml Leupeptin


Bioreagents and Chemicals:

Yeast Extract
α-Factor
2-Mercaptoethanol
Glucose or Dextrose
Peptone
Chymostatin
Pepstatin
Phenylmethylsulfonyl Fluoride (PMSF)
Bromophenol Blue
Nitrogen, Liquid
Glycerol
DMSO
Tris
Leupeptin
SDS
Acid-washed glass beads


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1292