Electroimmunochemical analysis of amphiphilic proteins and glycolipids stained with Sudan Black‐containing detergent micelles

Abstract

Micelles of non-ionic detergents can be colored through incorporation of the hydrophobic dye Sudan Black. This property can be used to identify amphiphilic antigens in complex mixtures. Thus, crossed immunoelectrophoresis with 0.05 % v/v Triton X-100 plus 0.002 % w/v Sudan Black shows selective staining of major amphiphilic proteins from human erythrocytes, thrombocytes and serum, from bovine milk fat globules and from yeast plasma membranes. The method may also give information about glycolipid antigens in detergent-solubilized membrane material, e. g. mannosyl-diinositolphosphoryl-ceramide from yeast. In this case, however, the membrane has to be solubilized with detergent plus Sudan Black and analyzed in detergent-free gels, where a stable micellar structure seems to exist. In a surplus of detergent, the glycolipid antigen may enter new micelles during electrophoresis, reducing the number of epitopes per micelle below two, thus avoiding precipitation. Analysis of purified lipopolysaccharide from Bordetellapertussis followed this pattern, whereas lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli, although heavily influenced by the presence of Triton X-100, were precipitated in the presence of a surplus of detergent. The described method is simple, fast and easy to perform, It requires neither extra equipment nor expensive chemicals. Sometimes heavily stained, detergent-containing precipitates of a non immunological nature may appear, but these can easily be discerned from immunoprecipitates.