Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Protein Chromatography
Protein chromatography on a cation-exchange cellulose column
Fractionation of blood serum proteins on a phosphocellulose column

A. Column preparation. An appropriate amount of phosphocellulose (P-Cellulose) is first washed three times with distilled Water by decantation, and then sequentially with: 0.5 M NaOH, distilled water, 0.1 M H₃PO₄, distilled water, and the starting buffer solution. Washing with distilled water is always continued until the pH of the supernatant reaches the pH of the water. The final equilibration of the ion exchanger with the starting buffer is performed directly in the column.

B. Chromatography. A 2 mL sample of serum, dialyzed for 24 hours against the starting buffer, is carefully applied to a 1x50 cm column using a pipette. Once the applied serum has entered the P-cellulose, any remaining residue is washed down with three 2 mL portions of the buffer solution. Protein fractionation is performed by step-gradient elution, applying the following solutions to the column: 0.02 M NaH₂PO₄ (starting buffer), 0.04 M phosphate buffer pH 5.8, 0.05 M phosphate buffer pH 6.2, 0.06 M phosphate buffer pH 6.6, 0.1 M phosphate buffer pH 7.2, and 0.1 M phosphate buffer + 0.5 M NaCl pH 9.5.

Elution is carried out at a flow rate of 50 mL/h, and the eluate is collected in 5–6 mL fractions. The optical density of the solution is monitored using an automatic instrument equipped with a chart recorder, such as an LKB Uvicord. Each subsequent eluting solution is applied only after the peak eluted by the previous one has completely emerged.