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The resin was then collected by centrifugation at 600 g for 10 min and washed in gravity flow column (Sangon Biotech) with five column volumes of buffer (pH 7.5) containing 20 mM HEPES, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl 2 , 1 mM MnCl 2 , 25 M TCEP, 0.1% (w/v) LMNG, 0.02% (w/v) CHS and 10 M ligand, followed by washing with fifteen column volumes of buffer (pH 7.5) containing 20 mM HEPES, 100 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl 2 , 1 mM MnCl 2 , 25 M TCEP, 0.03% (w/v) LMNG, 0.01% (w/v) glyco-diosgenin (GDN, Anatrace), 0.008% (w/v) CHS and 10 M ligand