2.5. Sephadex G-10 column chromatography of protein–copper
mixture
One milliliter of 50 mM Tris–HCl buffer (pH 8.4) containing
5 mM CuSO4 and 2.5 mg stylar proteins was loaded on a Sephadex
G-10 gel column (17 mmwidth × 250 mmlength) equilibrated with
50 mM Tris–HCl buffer. The elution was fractionated in 2 mL each,
then protein and copper ion concentrations of each fraction were
determined by the method of Bradford (1976) and atomic absorption
spectrophotometer (AA-6200; Shimadzu, Japan), respectively.