- 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.
- 1 mg/ml DNase and in water
- 100 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol
- 1M MgCl2
|1.||Resuspend the cells in chilled lysis buffer in a ratio of 1 g cell wet weight to 1 ml lysis buffer.
|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).|