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

Methods of Immunochemical Analysis
Protein Analysis by Gel Diffusion Methods
Immunogel Filtration

Principle of the method. The method combines the fractionation of the target protein using Thin-Layer Gel filtration with gel immunodiffusion analysis.

Applications. Characterization of the studied Proteins based on their molecular size and serological properties.

PROCEDURE

1. Thin-layer gel filtration. A layer of Sephadex G-100 or G-200 in 0.05 M veronal buffer, pH 8.4, is applied to an 8 x 20 cm Glass plate to obtain a layer thickness of 0.5 mm (see p. 231).

The plate is then mounted at the appropriate angle in a buffer reservoir filled with 0.02 M Tris-HCl, pH 8.0, containing 0.2 M NaCl. The Procedures for Sephadex equilibration and Sample application are described on p. 231. First, 1–2 µl of the test sample and control solutions are applied to the plate, then gel filtration is carried out; the plate is then removed from the apparatus and placed on a horizontal surface.

2. Immunodiffusion. The Sephadex gel is removed from the area between the Separation Zones of the test sample and control substances (Fig. 32). The entire surface of the plate is covered with a 1 mm layer of molten 1% Agar. After it solidifies, a longitudinal trough is cut between the zones of the test sample and control compounds and filled with specific antiserum. The plate is placed in a humid chamber, and the resulting precipitin bands are photographed in their Native State or after staining (Fig. 32).

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Fig. 32. Schematic diagram of immunogel filtration (see text for explanation).

NOTES

The main advantage of this method is that it allows the characterization of both the immunological properties and molecular size of a protein using very small sample amounts.