|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). |