Selective detection of terminally alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides by immunostaining on thin layer chromatograms.
A method for selective detection of terminally alpha 2-3 and alpha 2-6 sialylated neolacto-series gangliosides has been developed. The procedure involves separation of gangliosides on high performance thin layer chromatography plates, fixation of the silica gel, treatment with Vibrio cholerae neuraminidase and incubation of the plates with nLcOse4Cer-specific antibodies. Alkaline phosphatase-conjugated second antibodies were used to visualize bound first antibodies by generating a blue dye from 5-bromo-4-chloro-3-indolyl phosphate. Neolacto-series gangliosides from human granulocytes with terminally alpha 2-3 and alpha 2-6 linked N-acetylneuraminic acid served as examples. Neuraminidase treatment of gangliosides with alpha 2-3 substituted sialic acid is necessary prior to immunostaining whereas alpha 2-6 sialylated gangliosides can be detected without enzyme treatment. Steric hindrance of sialic acid bound in position 3 to terminal galactose prevented binding of the antibody to the Gal beta 1-4GlcNAc sequence whereas sialylation in position 6 to terminal galactose does not hinder recognition. The procedure is viable for the detection of amounts down to 10 ng of gangliosides. This method should be useful to screen gangliosides from different tissues or cell lines for the presence of such components, especially if only small quantities of material are available.
Müthing
J
J
Institut für Zellkulturtechnik, Universität Bielefeld, Germany.
Neumann
U
U
D000818:Animals; D002240:Carbohydrate Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002855:Chromatography, Thin Layer; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000737:chemistry; D005732:Gangliosides / Q000032:analysis* / Q000737:chemistry; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000494:pharmacology; D012794:Sialic Acids / Q000032:analysis
D005732:Gangliosides; D012794:Sialic Acids; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid