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Preparation of cell lysates from yeast using a French Press Preparation of cell lysates from yeast using a French Press


Equipment and reagents

Lysis buffer

50 mM Tris-HCl pH 7.5

50-200 mM NaCl*

1 mM EDTA, 5 mM DTT

1 mM PMSF

1 µg/ml leupeptin

1 µg/ml pepstatin A

*
 The NaCl concentration used in the lysis buffer depends fully on the application. In case of affinity chromatography on a Ni-column the NaCl concentration is usually 200 mM but when the first purification step is ion exchange chromatography no salt should be added.


Stock solutions

1 mg/ml DNase in water
10 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
1 mg/ml leupeptin in water
1 mg/ml pepstatin A in methanol
1 M MgCl2


Equipment

French Press cell (40 ml)



Procedure

1. Prechill the French Press cell at 4°C.
2. Resuspend the cells in chilled lysis buffer. Normally ratios of cell wet weight to buffer volume of 1:1 to 1:4 are used. Add 5 µl MgCl2 (1 M) and 1 µl DNase solution (1 mg/ml) per ml of cell suspension to avoid viscosity problems.
  • Add 10 µl PMSF (100 mM) per ml of celsuspension at this point.
3. Apply the sample to the French pressure cell and bring the cell under the desired pressure (7000 to 10,000 psi).
4. While maintaining the pressure, adjust the outlet flow rate to about one drop every second. Collect the cell lysate in a flask that is kept on ice.
5. Repeat steps 3 and 4 twice.
6. Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).