Specific staining on thin-layer chromatograms of glycosphingolipids of neolacto series and gangliosides with a terminal N-acetylneuraminyl residue by different procedures with wheat germ agglutinin.
Sensitive staining methods with wheat germ agglutinin were developed for the detection of glycosphingolipids of neolacto series (A) and gangliosides with a terminal N-acetylneuraminyl residue (B) on thin-layer chromatograms. (A) Neolacto series glycosphingolipids were treated by beta-galactosidase on the chromatograms in the presence of taurodeoxycholate. Then the chromatograms were incubated with biotinated wheat germ agglutinin followed by incubation with a complex of avidin and biotinated horseradish peroxidase, and the reaction was detected by 4-chloro-1-naphthol. In the case of gangliosides, sialidase treatment on the chromatograms was performed before the beta-galactosidase treatment. The sensitivity of the method for Lc3Cer, nLc4Cer, sialyl-nLc4Cer, and sialyl-nLc6Cer was 4 pmol, 7.6 pmol, 2.9 pmol and 1.4 pmol, respectively. (B) The gangliosides on the chromatograms were oxidized by periodic acid and reduced by NaBH4. Then the chromatograms were stained with wheat germ agglutinin as mentioned above. As little as 0.5 pmol of GM3, NeuAc-nLc4Cer, and NeuAc-nLc6Cer was detected by this method, whereas the detected limits for these gangliosides were 10 pmol, 10 pmol and 2 pmol, respectively, when periodate oxidation was omitted. GM4, GD3 and GD1a were an order less reactive than GM3, GM2, GM1 or GD1b were not stained under the same condition. In contrast to NeuAc-containing gangliosides, any gangliosides with N-glycolylneuraminic acid were not stained by the method in (B).
Higashi
H
H
Department of Pathology, Osaka University, Japan.
Sugii
T
T
Kato
S
S
D002855:Chromatography, Thin Layer / Q000379:methods; D005732:Gangliosides / Q000032:analysis*; D006028:Glycosphingolipids / Q000032:analysis*; D007202:Indicators and Reagents; D008832:Microchemistry; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000032:analysis*; D013194:Staining and Labeling; D013329:Structure-Activity Relationship; D014909:Wheat Germ Agglutinins
D005732:Gangliosides; D006028:Glycosphingolipids; D007202:Indicators and Reagents; D012794:Sialic Acids; D014909:Wheat Germ Agglutinins; D019158:N-Acetylneuraminic Acid