Detection of gangliosides of the GM1b type on high-performance thin-layer chromatography plates by immunostaining after neuraminidase treatment.
A method for the detection of GM1b-type gangliosides in complex mixtures of gangliosides was developed. The procedure involves separation of gangliosides on high-performance thin-layer chromatography plates, fixation of the silica gel, treatment with neuraminidase from Vibrio cholerae in the absence of detergent, and incubation of the plates with GgOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies are used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolylphosphate. The procedure is capable of detecting as little as 30 ng of gangliosides. Gangliosides from murine T lymphocytes and from human brain served as examples. Besides GM1b, GD1 alpha is also detectable by this method, whereas the human brain gangliosides GM1a, GD1a, GD1b, and GT1b are not, because they are neuraminidase resistant. Since terminally sialylated gangliosides such as GM1b were described as virus receptors, and certain other terminally sialylated gangliosides are discussed as tumor markers, this method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small amounts of material are available.
Müthing
J
J
Gesellschaft für Biotechnologische Forschung mbH Braunschweig, Federal Republic of Germany.
Mühlradt
P F
PF
D000818:Animals; D001923:Brain Chemistry; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D005677:G(M1) Ganglioside / Q000145:classification / Q000302:isolation & purification*; D005732:Gangliosides / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009439:Neuraminidase; D013601:T-Lymphocytes / Q000032:analysis; D014734:Vibrio cholerae / Q000201:enzymology
D005732:Gangliosides; D005677:G(M1) Ganglioside; D009439:Neuraminidase