This is a cached page for the URL (http://www.embl-heidelberg.de/ExternalInfo/geerlof/draft_frames/protocol_database/Purification/ec_Ecoli_enzyme.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Preparation of cell lysates from E 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).