Superdex hiload s75 16 600 column

N-terminally 6xHis-tagged E. coli RF1 was overexpressed in BL21 E. coli cells grown at 37°C from overnight culture in LB medium and in presence of 100 μg/mL Ampicillin. Protein expression was induced at A₆₀₀ of 0.4 by adding IPTG to a final concentration of 1 mM. RF1 was expressed from pET28-plasmid kindly provided by Rachel Green (John Hopkins University, Baltimore). After 1 h of expression, cells were lysed using a microfluidizer. The cell lysate was cleared by centrifugation in a SS34 rotor (Sorval) at 4°C and 44,100 x g for 30 min. Purification of His-tagged RF1 was done with Protino Ni-NTA agarose beads (Macherey-Nagel). The final eluate was applied onto a Superdex HiLoad S75 16/600 column (GE Healthcare) to yield the final concentrated protein in gel filtration buffer (50 mM HEPES pH 7.4, 50 mM KCl, 100 mM NaCl, 2% glycerol and 5 mM 2-mercaptoethanol).