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

Protein Chromatography
Isolation of IgG from blood serum using batch fractionation on DEAE-Sephadex

Under appropriate conditions, DEAE-Sephadex binds all Serum Proteins except for the IgG fraction. Therefore, serum protein fractionation using this ion exchanger is highly convenient for isolating immunochemically pure IgG, and is also useful for the further purification of IgG preparations obtained by other Methods.

THE PRINCIPLE OF the batch method is as follows. The serum to be fractionated is mixed with DEAE-Sephadex equilibrated with an appropriate buffer solution. The ion exchanger binds almost all serum proteins, leaving IgG in the solution, which is easily separated by washing. Mixing with DEAE-Sephadex is repeated twice (each time with a fresh portion of the ion exchanger), yielding an IgG preparation free from contamination by other serum proteins, as confirmed by immunoelectrophoretic analysis. Since the IgG fraction does not bind to the ion exchanger at all, proteins of this Class can be isolated almost quantitatively in their Native State without risk of Denaturation, which is a major advantage of this method over other IgG isolation techniques.

Procedure

DEAE-Sephadex is treated in the same manner as for Column Chromatography, and then equilibrated with 0.01 M phosphate buffer, pH 6.5. Once equilibration is complete, the buffer is removed as much as possible so that the ion exchanger remains barely moist. This is most conveniently done by placing the DEAE-Sephadex on a Büchner funnel lined with filter paper and drawing the buffer through under vacuum using a Water aspirator.

The serum to be fractionated (1–30 ml) is poured into a beaker, the required amount of wet ion exchanger is added, and the mixture is stirred to obtain a homogeneous suspension. The mixture is left at 4°C for 1 h and then transferred onto filter paper lining a Büchner funnel. The gel is washed with cold 0.01 M phosphate buffer, pH 6.5, until protein is no longer detectable in the eluate (by reaction with sulfosalicylic acid). The collected buffer containing the protein is mixed with a fresh portion of DEAE-Sephadex, the resulting suspension is again left at 4°C for 1 h, and the unbound protein is washed out as described above. The wash buffer, which now contains only pure IgG, can be lyophilized or concentrated by pressure dialysis.

Recommended reading

Determann H., Gel chromatography, Springer Verlag, Berlin, Heidelberg, New York, 1968. (H. Determann, Gel Chromatography, Mir Publishers, Moscow, 1970).

Leach S. J., Physical Principles and Techniques of Protein Chemistry, Academic Press, New York, London, 1969.

Williams C. A., Chase M. W., Methods in Immunology and Immunochemistry, Vol. II, Academic Press, New York, London, 1968.