Hiprep 26 60 sephacryl s 200 column

Cell pellets were thawed and resuspended with Lysis Buffer comprising 50 mM Tris pH 8.0, 0.4 M NaCl, 5 mM β-mercaptoethanol and 200 μg/mL lysozyme. One tablet of EDTA-free protease inhibitor (Roche Applied Science) was added per 10 mL of cell lysis buffer. After 20 min incubation on ice the cell suspension was disrupted by sonication (Qsonica 125 Ultrasonic Processor). Cell debris was removed by centrifugation at 20K × g for 25 minutes. The soluble lysate was filtered with a 0.45μm filter and added to a nickel column (PerfectPro matrices) equilibrated with buffer A (50 mM Tris pH 8.0, 0.4 M NaCl, 5 mM imidazole and 5 mM MgCl₂). Protein was eluted with buffer A supplemented with 250 mM imidazole. Fractions containing enolase were identified by SDS-PAGE gel, pooled and concentrated using a Centricon filter unit (Millipore). The protein sample was then subjected to a HiPrep 26/60 Sephacryl S-200 column (GE Healthcare) equilibrated with 25 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl₂ and 0.5 mM DTT. The predominant protein peak eluted with an apparent molecular weight of 93 kDa which is consistent with the native molecular weight of dimeric E. coli enolase (91.2 kDa). Appropriate fractions were pooled, concentrated to 8.0 mg/mL, flash frozen with liquid nitrogen and stored at −80 °C. The high level of protein purity was confirmed by SDS-PAGE stained with Coomassie Blue.