|1. ||Prepare the glass beads by washing them in concentrated HCl, followed by extensive rinsing (check that then pH is neutral) and drying. The dried beads should be chilled before use. |
|2. ||Resuspend the cells in an equal amount of chilled lysis buffer and place the suspension in a sturdy tube (preferably not glass). |
- Add the PMSF (10 µl PMSF (100 mM) per ml of celsuspension) at this point.
|3. ||Add 1 - 3 g of chilled glass beads per gram of cell wet weight. |
|4. ||Vortex 3 - 5 times for 1 minute, each time keeping the cells on ice for 1 minute between vortexings. use the highest setting of the vortex mixer. |
|5. ||Remove the glass beads. |
|6. ||Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman). |