Purification of Ecoli-Muts

Transform E.coli strain BL21 (DE3) with cloned E.coli MutS (e.g. pET30b) .

Induction.

Spin down cells.

Resuspend in  lysis buffer (20 mM Tris–HCl, pH 8.0, 0.5 mM 2-mercaptoethanol, 1 mg/ml lysozyme and 250 mM PMSF)

lyse by sonication.

Load to 5~10 ml Hi-trap Ni-column pre-equilibrated with binding buffer (20 mM Tris–HCl, pH 8.0, 0.5 mM 2-mercaptoethanol and 500 mM NaCl).

Wash the column with 5 mM imidazole

Elute with 300 mM imidazole.

Load to Q HP or MonoQ column equlibrated with buffer  (20 mM Tris–HCl, pH 8.0, 1 mM EDTA and 1 mM DTT).

Elute with linear gradient