CM-Sepharose column chromatography with β-mercaptoethanol (a reducing agent) of agkaggregin. The protein fraction containing agkaggregin was desalted and ultrafiltered to a volume of 20 ml and then applied to a CM-Sepharose column (1.4 × 40 cm) pre-equilibrated with 20 mM sodium citrate pH 5.0 at a flow rate of 120 ml h−1. The column was sequentially eluted with 200 ml solution I (20 mM sodium citrate pH 5.0), 500 ml solution II (20 mM sodium citrate solution pH 5.6) and 300 ml solution III (20 mM sodium citrate pH 5.6 containing 10 mM β-mercaptoethanol). The column was further washed with 800 ml of NaCl solution with a linear gradient from 0 to 0.5 M. The elution was monitored at 280 nm. The absorbance at 280 nm and the elution of the salt gradient are shown by a solid line and a dashed line, respectively. The last effluent peak, indicated by a solid bar, contains the protein agkaggregin.