| Abstract: | Methods for direct immunological identification of single protein components after fractionation of a protein mixture in microgels are described. Protein mixtures were separated with high resolution in polyacrylamide microgradient gels and transferred after electrophoresis into agarose layers containing suitable antisera. Monospecific as well as polyvalent antisera were used. The formation of immunoprecipitates could be observed within approx. 1 h. Immunoprecipitates are also formed in the presence of sodium dodecylsulfate or other detergents, thus allowing immunoreactions to be performed with water-insoluble proteins. Staining of the proteins in the gels did not completely inhibit the immunoreaction, while dansylation of proteins had no effect. The influence of different detergents e.g. sodium dodecylsulfate, Triton X-100, Brij 99, np-40 and urea, as well as different reducing agents e.g. mercaptoethanol, dithiothreitol, thioglycolic acid, on two-dimensional microdiffusion was also studied. When suitable concentrations of these compounds were used, the formation of immunocomplexes was observed within approx. 1 h. This technique can also be applied to immunoreactions with water-insoluble proteins dissolved in detergents. |
