Preparation of cell lysates from E. coli by enzymatic lysis
Materials
Chemicals
lysosyme
Lysis buffer
50 mM Tris-HCl pH 7.5
50-200 mM NaCl*
5% glycerol (v/v)
1 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 solutions
1 mg/ml DNase and in water
100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
1M MgCl2
Procedure
1.
Resuspend the cells in chilled lysis buffer in a ratio of 1 g cell wet weight to 1 ml lysis buffer.
Add the PMSF (10 µl PMSF (100 mM) per ml of celsuspension) at this point.
2.
Add lysosyme to a final concentration of 300 µg/ml and incubate the cell suspension at 4°C for 4 h.
3.
Add 5 µl MgCl2 (1 M) and 1 µl DNase solution (1 mg/ml) per ml of cell suspension and incubate the solution at 4°C for 30 min.
4.
Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).
