Purification of Ecoli-Muts
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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