| Contributor: |
The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
|
| |
1. Grow a 2 liter yeast culture in YPD to an optical density at 600 nm (OD600) of 1.5 to 2.0 (see Hint #2)
2. Centrifuge at 1,500 X g to pellet the cells and discard the YPD.
3. To the cell pellet add 30 ml of cold ddH2O and mix well.
4. Centrifuge at 1,500 X g to pellet the cells and discard the ddH2O.
5. To the cell pellet add 30 ml of cold Extraction Buffer and mix well.
6. Centrifuge at 1,500 X g to pellet the cells and discard the Extraction Buffer.
7. Pre-cool a large (approximately 8 inch diameter) mortar and pestle by placing on dry ice and adding a small amount of liquid nitrogen.
8. Weigh the cell pellet (see Hint #3).
9. Place the entire cell pellet into a 10 ml syringe and push the cell pellet into a 50 ml tube suspended in liquid nitrogen (see Hint #4).
10. Place the frozen yeast pellet into the pre-cooled mortar and grind the larger pieces for 1 min.
11. Add a small amount of liquid nitrogen and grind again for another 1 min. Repeat this step one more time (see Hint #5).
12. Using a pre-cooled spatula, scrape the powder into a pre-cooled weigh boat and dump the entire power into a 50 ml conical tube (see Hint #6).
13. Transfer the tube to wet ice and 15% (v/w) of Extraction Buffer (compared to the total cell weight, i.e. 5 g cells + 750 μl buffer) and incubate on ice 20 min.
14. Scrape the yeast sorbet into an ultracentrifuge tube and centrifuge in ultracentrifuge at 100,000 X g for 1 hr at 4°C (33,000 rpm in an SW55 rotor).
15. After centrifugation, approximately one-third of the volume is the cell pellet and two-thirds of the volume is a clear yellow liquid with a thin white lipid layer on top. Remove the clear liquid with a syringe being careful to avoid the pellet and the lipid layer.
16. Dialyze the clear solution 3 times for 1 hr against 500 ml Dialysis Buffer.
17. Aliquot the dialyzed solution and freeze in aliquots of appropriate volume.
|
| Extraction Buffer |
| 1X Leupeptin*
1X Benzamidine*
1X Chymostatin*
245 mM KCl
100 mM HEPES, pH 7.9
*Add just before use
2 mM DTT*
1 mM EDTA
5 mM EGTA
1X Pepstatin*
|
 |
| Chymostatin (2500X) |
| in DMSO
5 mg/ml Chymostatin
Store at -20°C
|
|
| Leupeptin (500X) |
| Store at -70°C up to 6 months
in Ethanol
0.15 mg/ml Leupeptin
|
|
| Dialysis Buffer |
| 1X Leupeptin*
1X Benzamidine*
1X Chymostatin*
100 mM KCl
5 mM MgCl2
*Add just before use
2 mM DTT*
20% (v/v) Glycerol
1 mM EDTA
20 mM HEPES, pH 7.9
1X Pepstatin*
|
|
| YPD Media |
| 10 g/liter Yeast Extract
20 g/liter Glucose
20 g/liter Peptone
Autoclave for 20 min, cool to room temperature
|
|
| Pepstatin (200X) |
| in Methanol
Store at -20°C
0.28 mg/ml Pepstatin
|
|
| Benzamidine (100X) |
| Store at -20°C
31 mg/ml Benzamidine
|
|
| PMSF |
| 0.1 M PMSF (CAUTION! see Hint #1)
Store at -20°C
in Ethanol
|
|
|
| |
HEPES
Glucose
Nitrogen, Liquid
EDTA
Leupeptin
Chymostatin
PMSF
Pepstatin
Ethanol
Potassium Chloride
EGTA
Yeast Extract
Glycerol
Methanol
DMSO
Peptone
Benzamidine
Magnesium Chloride
|
| |
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. This can be achieved by adding a 1.5 ml saturated overnight yeast culture to 1 liter of YPD and growing approximately 14 hr at 30°C.
3. The final weight of cells should be between 5 to 6 grams.
4. This cell pellet can be frozen at -70°C for at least one year with no loss of activity.
5. The grinding process should continue until the entire material is a fine powder.
6. If preparing multiple yeast extracts the sample can remain on dry ice until all the samples are ready for the next step.
|
