Adsorption and Elution on Calcium Phosphate Gel

Prepare the gel by mixing 500 ml of 1 M calcium chloride and 500 ml of IM dibasic sodium phosphate. Wash the gel on a glass filter with 1000 ml of ice-cold water and then suspend in 500 ml of 

A column of CM-Sephadex (2.5x20 cm, Pharmacia Biotech, Uppsala, Sweden) is prepared according to the manufacturer s instruction by using 10 mM PB, pH 6.0, as an equilibration buffer. Apply the dialyzed fraction to the column, wash with 200 ml of 10 mM PB, pH 6.0 (this percolate contains component I), and elute component II with a gradient from 0 to 0.4 M sodium chloride in 1000 ml of 10 mM PB, pH 6.0. Collect the fractions containing component II and concentrate by ultrafiltration through a P0200 membrane (UHP-43K, Advantec Co., Tokyo, Japan). 

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