Diffusion of Proteins through Lipophilic Agar Gels

IF agar is used as a gelling agent, the aqueous phase of the gel should not be reduced below 38 per cent, otherwise the gel loses quickly its homogeneity and the necessary rigidity for handling it experimentally. Thus the intermediate gels, through which the diffusion occurs, contain 2 per cent agar (‘Bacto-Agar’, Difco Laboratories), 38 per cent aqueous buffer solution and 60 per cent of a lipophilic component. In gels Nos. V, VI and VII the latter consists of 48 per cent 1,2-propandiol and 12 per cent (by weight) of a further lipophilic component as indicated in Table 1. These additions raised the lipophilic properties considerably. The gel is formed by pouring 11 ml. of the hot, thoroughly mixed solution on a hot glass plate, such as are used in the ‘Elphor’ paper strip scanner. After setting, the gel is detached from the glass plate and laid upon the basis gel of 1 per cent agar in buffer. Drops of 2 per cent solutions of pure serum protein fractions, containing 200 µgm. of protein, are now placed, using an ‘Agla’ pipette, at regular intervals on the surface of the intermediate gel. After 48 hr. in a wet chamber the proteins having diffused into the basis gel are measured by a technique which I have already described 1,2 (Fig. 1). The following protein-fractions were used: serum albumin (mol. wt. 69,000); α 1 -lipoprotein (mol. wt. approx. 250,000, ref. 3, 45 per cent lipoid-component, ref. 4; Behringwerke, Marburg/Lahn) and α 2 -macroglobulin (mol. wt. 846,000, ref. 5; Behringwerke, Marburg/Lahn). Fig. 1 shows results of the standardized technique, allowing 7 diffusion tests per glass plate. If the intermediate gel contains only agar, average values should not differ by more than 5 per cent; with a lipophilic component in the gel the limit is rather 8 per cent. Microphotograms have shown that the latter gels are less homogeneous.