The insoluble fraction of the lysate was
removed by centrifugation at 47,000 g for 30 min at 4 C, and the
supernatant was filtered through a 0.2 mm pore-size filter and then
loaded onto a Talon Superflow resin (Clontech) equilibrated in
phosphate buffer (50 mM, pH 7) containing 300 mM NaCl and
10 mM imidazole to improve the interaction specificity in the affinity
chromatography step.