Preparation of cell lysates from E. coli by enzymatic lysis

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℃ 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℃ for 30 min.4.Remove cell debris byμltracentrifugation at 4℃ for 30 min at 45 000 rpm using a 45Ti rotor (Beckman).