Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill

The following technical report Making Whole Cell Extract of Saccharomyces cerevisiae cells using the Retsch MM301 Ball Mill was published on 24. August 2005. Courtesy of the Staley Lab at the University of Chicago. Download as PDF file.

Cell Preparation

Harvest 2 liters of cells grown to mid to late log phase by spinning at 6,000g for 15 minutes. Wash cells twice with chilled buffer AGK. Estimate volume of cell pellet and add 0.4 volumes chilled AGK. Vortex to make a thick cell suspension. Add protease inhibitor cocktail to a final concentration of 1x. 

Add the suspension dropwise into a 50 ml conical that is filled with liquid nitrogen, using a pipette or a syringe fitted with a needle. Leave a few seconds between drops so they freeze properly. Refill the liquid nitrogen as necessary and remember to puncture the cap several times with a needle or it will explode! The frozen pellets can then be stored at -80°C until you want to continue.

Milling the Extract

Before running the mill, fill the canisters with some water (no ball! Running the canisters with the ball, but without a solid sample will ruin the canister) and run the mill at 30 Hz briefly to make sure the Teflon O ring in the canister is still creating an effective seal. If the canisters leak, replace the O ring before proceeding.

Pre-chill the stainless steel grinding canisters and the grinding balls in liquid nitrogen. Use either the 25 mm or 30 mm ball depending on the amount of cell pellets, if the canister is too full, the sample will not be ground effectively. Typically, a 2L harvest results in a cell pellet of approximately 5 ml, plus 2 ml of AGK buffer. The 7 mls of frozen cell suspension fit comfortably with the 30 mm ball in the 50 ml grinding canister (cells will fill most of the bottom half of the canister, Fig. 1). If only one sample is being milled, use the second empty canister (without a ball) as a balance.

_{Figure 1}

_{Figure 2}

Harvest 2 liters of cells grown to mid to late log phase by spinning at 6,000g for 15 minutes. Wash cells twice with chilled buffer AGK. Estimate volume of cell pellet and add 0.4 volumes chilled AGK. Vortex to make a thick cell suspension. Add protease inhibitor cocktail to a final concentration of 1x. 

Add the suspension dropwise into a 50 ml conical that is filled with liquid nitrogen, using a pipette or a syringe fitted with a needle. Leave a few seconds between drops so they freeze properly. Refill the liquid nitrogen as necessary and remember to puncture the cap several times with a needle or it will explode! The frozen pellets can then be stored at -80°C until you want to continue.

Processing the Powdered Cells for Whole Cell Extract

Thaw the powder rapidly and completely (with manual agitation) in a 25°- 30° bath and then place on ice. Spin 17K 30 minutes in an SS34 rotor at 4°. Avoiding the pellet and the lipoprotein layer at the top of the tube, transfer the supernatant into an ultracentrifuge tube. Spin at 100,000g for 1 hour. Again, avoiding the lipoprotein layer and the pellet, remove the middle layer. Dialyze twice in tubing with a molecular weight cutoff of 10,000 for 1.5 hours at 4°C against 2 L of Buffer D (procedure reviewed in Stevens and Abelson, 2002.) Spin extract at top speed in microfuge at 4° for 5 minutes to remove sediments and precipitates. Aliquot and freeze supernatants in liquid nitrogen. Store at -80°C.

_{Figure 3. Extracts made from cells disrupted by the Retsch MM301 Ball Mill are comparable to those made by cells disrupted by mortar and pestle. The same yeast strain was processed in parallel with the only difference being the method of cell disruption. Cells were disrupted either by hand with a mortar and pestle or by five 3 minute cycles in the Retsch MM301 Ball Mill set at either 10, 20 or 30 Hz. A. Activity of yeast extract was measured by efficiency of splicing an in vitro transcribed actin pre mRNA substrate (reviewed by Stevens and Abelson, 2002). B. A 1:10 dilution of cells was viewed under a 100X objective immediately after disruption. All of these cell samples started at roughly the same concentration. The apparent difference in cell abundance in the 20 and 30 Hz samples compared with the 10 Hz sample is due to aggregation of the cell debris.}

Notes and Critical Parameters

1. Splicing activity of extracts made in parallel from cells disrupted by mortar and pestle versus the ball mill (at 10 Hz) have comparable splicing activity (Fig. 3A), indicating that RNA, as well as protein, remains intact and active after processing with the ball mill. Increasing the cycles of disruption from 5 cycles to 8 or 10 cycles did not increase the efficiency of splicing (data not shown). Cell disruption by the ball mill is faster, less labor intensive and more reproducible than cell disruption by mortar and pestle.
2. Extracts made from cells disrupted at higher than10 Hz show reduced splicing activity (Fig. 3A). However, other yeast labs have reported using the ball mill at 30 Hz for immunoprecipitation (see protocol from Marcus Kalkum, available from Retsch). It is advisable to try a range of milling times and speeds for new applications.
3. Note that the extract in Fig. 3A made from cells milled at 20 Hz shows a tighter mRNA band, which may be due to reduced poly A activity compared to the other extracts. This trend did not reproduce in a comparable trial with a different strain. The observed differences in mRNA levels in Fig. 3 suggest that the varied methods of cell disruption may generate extracts with slightly different biochemical properties.
4. Disruption of cells at 10 Hz is incomplete, but looks comparable to disruption by mortar and pestle (Fig. 3B). While most cells are disrupted when milled at 30 Hz, extract made from these cells is inactive. The debris from cells milled at 30 Hz tends to stick together and may result in the pelleting of complexes, such as the spliceosome. Therefore, milling higher than10 Hz may not be advisable for other procedures.
5. When comparing extracts made from cells disrupted by mortar and pestle versus the ball mill, we saw comparable levels of protein, as assayed by BioRad Protein Assay with a BSA standard, and comparable levels of spliceosomal snRNAs, as assayed by primer extension (data not shown). We found the extracts behaved comparably when depleted and reconstituted for a spliceosomal protein and a spliceosomal snRNA (data not shown).

Buffers and Solutions

Protease Inhibitor Cocktail:

• 100mM AEBSF
• 100 ug/ml Leupeptin
• 100 ug/ml Aprotinin
• 100 mM Benzamidine

Buffer AGK:

• 10 mM HEPES pH 7.9
• 1.5 mM MgCl₂
• 200 mM KCI
• 10% vol/vol glycerol
• 0.5 mM DTT (added fresh before use)

Buffer D:

• 20 mM HEPES pH 7.9
• 0.2 mM EDTA
• 50 mM KCI
• 20% vol/vol glycerol
• 0.5 mM DTT (added fresh before use)

References

Stevens SW, Abelson J. Yeast pre-mRNA splicing: methods, mechanisms, and machinery. Methods Enzymol. (2002) 351:200-20

^{Created on August 24th, 2005}