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After centrifugation at 20,000 g for 1 h, supernatants were incubated with Ni 2+ Sepharose and applied to two successive washes with buffer A (50 mM Tris pH 7.5, 1.2 M KCl, 20 mM imidazole) and buffer B (50 mM Tris pH 7.5, 150 mM NaCl)

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