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

Gel Filtration of Proteins and Peptides
Fractionation of Serum Proteins by Gel Filtration

Principle of the method. Serum protein fractions are separated on a Sephadex G-200 Column according to their molecular weight.

Scope of application. Fractionation of Protein mixtures and purification of isolated protein fractions.

PROCEDURE

1. Preparation of the Sephadex G-200 column. Column size is 3x80 cm. Sephadex G-200 is suspended in 0.1 M Tris-HCl pH 8.0 containing 0.2 M NaCl and allowed to swell for 24 hours. Fine particles floating in the supernatant are removed by decantation. A paper filter or Glass wool is placed at the bottom of the column, and the column is filled to 1/3 of its height with buffer solution. First, a portion of Sephadex G-25 is introduced into the column to form a 2 cm layer (Sephadex G-25 must be previously allowed to swell in the buffer solution for 24 hours). Then, the column is filled with a dilute suspension of Sephadex G-200, and after a few minutes of settling, the outlet is opened. As the buffer solution flows out, Filling of the column with the Sephadex suspension is continued, ensuring that air bubbles formed during filling do not get trapped in the gel bed. Once the gel bed reaches the required height, one volume of buffer solution is passed through the column.

2. Serum application. The column outlet is opened, and the buffer solution is allowed to drain until its level at the top of the column reaches the gel bed surface. Then, 4–5 ml of serum, previously dialyzed against the buffer solution for 1 h, is carefully layered onto the Sephadex, taking care not to disturb the top layer of the gel. Once the applied sample has entered the gel, any remaining residue is washed down with three 4 ml portions of the buffer solution.

3. Elution. For elution, a total of 700–800 ml of buffer solution is passed through the column at a flow rate of 40–60 ml/h, and 5 ml fractions are collected. The optical density of the eluate is monitored either continuously using a “Uvicord”, or by spectrophotometry of each individual fraction at 280 nm.

NOTES

1. Serum Proteins are eluted from the Sephadex G-200 column in three distinct fractions. The first fraction (corresponding to the first peak) contains high-molecular-weight proteins, such as macroglobulin and Lipoproteins. The second fraction (corresponding to the second peak) contains proteins with molecular weights close to IgG, namely IMMUNOGLOBULINS G and A. In the third fraction (corresponding to the third peak), lower-molecular-weight proteins, such as albumin and transferrin, are eluted. During the fractionation of sera from patients with IgG- or IgA-type myelomas, the second peak becomes very large. An increase in the first peak is characteristic of Waldenström's macroglobulinemia.

2. To protect the top layer of the gel from disturbance during Sample application, a disc of filter paper can be placed on the gel surface. In Gel filtration on Sephadex G-75, G-100, G-150, and G-200, especially when fractionating proteins, it is advisable to leave a small amount of buffer solution in the column above the Sephadex and apply the sample beneath it. This is feasible if the density of the applied sample is higher than that of the buffer solution. The density of the protein solution to be fractionated can be increased by adding an appropriate amount of sucrose.

3. Typically, gel filtration is continued until all proteins are eluted from the column; therefore, washing the column after the run may not be necessary. However, it is still recommended to pass several volumes of buffer solution through the column before each fractionation.

4. If the column is intended to be used over a long period, it is advisable to suspend the Sephadex in a buffer solution containing merthiolate, phenol, or toluene to prevent bacterial growth. Sephadex types can be autoclaved (at 110°C for 40 min) without altering their properties.

5. Once the Sephadex gel has swollen and settled, one should

carefully remove the fine particles floating in the supernatant, as they can reduce the flow rate of the eluent through the column.    

6. Elution can also be performed with a 0.1 M Tris-HCl pH 8.0 solution containing 1 M NaCl.