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Preparation of cell lysates from E Preparation of cell lysates from E. coli by sonication


Materials

Lysis buffer

50 mM Tris-HCl pH 7.5

50-200 mM NaCl*

5 mM DTT

1 mM PMSF

*
 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 solution

100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol



Procedure

 
1. Resuspend the cells in chilled lysis buffer. Normally ratios of cell wet weight to buffer volume of 1:1 to 1:4 are used.
2. Cool the cell suspension on ice for 10 min.
  • Add 10 µl PMSF (100 mM) per ml of celsuspension after this step. 
3. Sonicate the cell suspension with 10 short burst of 10 sec followed by intervals of 30 sec for cooling.
  • Keep the suspension at all times on ice.
  • Avoid foaming.
  • Don’t go away while the sonicator is in operation. It is possible that the beaker breaks or turns in the melting ice.
4. Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).