Chromatogram of one-step gradient elution using column 2. In this separation, 13 mg of protein were loaded onto the 75 mL column. Binding buffer contained 0.6 M (NH4)2SO4. Protein P34 was eluted using 0.25 M (NH4)2SO4. Subsequently, still bound protein at the column was eluted with phosphate buffer containing no (NH4)2SO4.