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

Gel Filtration of Proteins and Peptides
Isolation of IgG from serum by batch fractionation on DEAE-Sephadex
Desalting of protein solutions on a Sephadex column

Principle of the method. As low-molecular-weight substances, salts present in protein solutions penetrate the gel particles during Gel filtration, which retards their migration through the gel. Meanwhile, due to their high molecular weight, Proteins pass through the Sephadex gel Column unhindered, emerging with the eluate front.

PROCEDURE

1. Column preparation. To desalt 100 ml of protein solution, approximately 25 g of dry Sephadex G-25 is required. The volume of the solution to be desalted must not exceed one-fifth of the gel bed volume.

Dry Sephadex is suspended with stirring in 0.05 M Tris buffer, pH 7.2. After the gel particles settle, the supernatant (which may sometimes be turbid) is decanted. Resuspension in fresh portions of the buffer and settling are repeated until the supernatant is completely clear. The suspension is then poured into the column, allowed to settle for a short time, and then the column outlet is opened to drain the buffer until its level reaches the top of the Sephadex bed.

2. Desalting. The protein solution is passed through the Sephadex gel. The protein content in the desalted fractions is determined either by absorbance at 280 nm or by precipitation with trichloroacetic acid.

NOTES

1. Desalting is a form of Group Separation where the compounds being fractionated are eluted in two fractions. One fraction (in this case, high-molecular-weight compounds—proteins) elutes in the void volume. The other, containing low-molecular-weight compounds, penetrates the gel particles and thus emerges from the column significantly later during elution, well separated from the first.

2. In group separation, high-molecular-weight compounds are not fractionated. In this case, gel filtration, like dialysis, is used to separate proteins from low-molecular-weight impurities and can be used as an alternative to it. Protein desalting can be performed on Sephadex G-25 and G-50, as well as on Bio-Gel P6 and P10. For peptide desalting, Sephadex G-10 and G-15 or Bio-Gel P2 and P4 should be used.

3. For group separation, it is practical to use short chromatographic columns 20–30 cm in length. The bed volume of the gel must be at least 4 times the volume of the solution being desalted.