Washing Buffer: 20 mM Tris pH 8.0, 250 mM NaCl, 5% Glycerol, 50 mM imidazole
Elution Buffer: 20 mM Tris pH8.0, 250 mM NaCl with 5% glycerol, 250 mM imidazole
The crude extract was cleared by centrifugation. The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of washing and the protein was eluted with 25ml elution.
The protein was further purified to homogeneity by Superdex200 column (26x60) (Amersham Biosciences), equilibrated with 20 mM Tris buffer, pH 8.0, and 150 mM NaCl, at flow rate 4 ml/min.
TEV protease was added to combined fractions containing NLE1 protein overnight at 4°C.
The protein was loaded onto ion-exchange chromatography on Source 30 Q column, equilibrated with buffer 20mM Tris pH7.5, and eluted with linear gradient of NaCl up to 1 M concentration (20CV).
Protein yield: 34 mg/L