Cupric Chloride → beta-Cyclodextrin copper

Participants

Reaction Steps

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.

Provenance & Evidence