Cupric Chloride → beta-Cyclodextrin copper

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The beta-cyclodextrin-copper biaffinity column was prepared by mixing 0.5 ml each of the sulfated beta-cyclodextrin polymer and chelating Sepharose (Pharmacia) that had been saturated with copper (II) chloride. A sample (16 ml) of Chondrosarcoma-derived growth factor partially purified by batchwise adsorption to heparin-Sepharose® was applied directly to this biaffinity column (1×1.3 cm) pre-equilibrated with 2M NaCl, 10 mM Tris, pH 7. The column was rinsed consecutively with 40 ml each of the following four reagents in 10 mM Tris, pH 7: (i) 2M NaCl, (ii) 0.6M NaCl, (iii) 0.6M NaCl plus 10 mM imidazole, and (iv) 0.6M NaCl. Finally, the growth factor was eluted at a flow rate of 20 ml/hour with a linear NaCl/imidazole gradient from 100 mM imidazole in 10 mM Tris, pH 7. Fractions (4 ml) were collected, monitored for conductivity (Radiometer American conductivity meter) and assayed for growth factor activity.

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