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

Protein Chromatography
Protein Chromatography on a DEAE-Cellulose Column
DEAE-cellulose in the Cl- form

a) Conversion of DEAE-Cellulose to the Cl^- form. DEAE-cellulose in the OH^- form, treated as described above and suspended in distilled Water, is stirred for 15 min at room Temperature with 3 volumes of 0.05 M Tris-HCl, pH 8. The suspension is allowed to settle, and after decanting the supernatant, this stirring process is repeated twice. Then, the DEAE-cellulose is suspended in the same buffer solution and packed into a Column of appropriate dimensions so that the ion-exchanger bed is homogeneous and free of air bubbles. After packing the column, the DEAE-cellulose is washed with the aforementioned buffer solution until the electrical conductivity and pH of the eluate match those of the washing solution. At this stage, the column is ready for fractionation.

b) Chromatography of native Proteins on DEAE-cellulose in the Cl^- form. The material to be fractionated is dissolved in a small volume of the starting buffer solution (typically 100 mg in 3—5 ml). The solvent is a 0.05 M Tris-HCl buffer solution, pH 8 (the specified molarity refers to Cl^- ions), in which the material binds to the ion exchanger. The solution of the material to be fractionated is previously dialyzed against the same buffer solution for 10 h.

Carefully remove the buffer solution from the top of the column above the cellulose so that a liquid layer of no more than 2—3 mm remains above the ion exchanger. The solution of the material to be fractionated is gently layered onto the DEAE-cellulose using circular hand motions, touching the curved tip of the pipette to the inner surface of the column. During layering, It is important not to disturb the DEAE-cellulose particles. Once the applied solution has entered the ion exchanger, the remaining material is washed from the inner walls of the upper column three times with 0.5—1.0 ml of the 0.05 M buffer solution.

At a 0.05 M concentration of Cl^- ions, native proteins bind well to DEAE-cellulose. Unbound Components of the fractionated mixture are removed by washing the column with one volume of the buffer solution.

c) Gradient elution. If fractionation is performed on a 1.5x35 cm column, 100 ml vessels are sufficient for elution, one serving as a mixer and the other as a reservoir for the buffer solution. A 0.1 M buffer solution is poured into the mixer, and a 0.5 M buffer solution is poured into the reservoir; they are connected by a Teflon or polyvinyl chloride tube free of air bubbles, and the mixer, placed on a magnetic stirrer, is connected to the column.

Using a screw clamp on the Teflon or polyvinyl chloride tube at the column outlet, the optimal flow rate of the liquid is set to approximately 1 ml/min.

The eluate is collected in 0.5—1.0 ml fractions. If an appropriate instrument is available, continuous spectrophotometry and recording of the optical density of the eluate are performed, and the collected fractions are pooled According to the chart plotted by the recorder. In the absence of an automatic flow-through spectrophotometer, all eluate fractions are measured spectrophotometrically at 280 nm, an optical density profile is plotted, and the desired fractions are selected based on it.

By determining the absorbance of the starting material and the obtained fractions, the yield of the preparation resulting from the chromatographic Separation can be established.