Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase.

Abstract:

:A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccharides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4Cer, II3NeuGc-GgOse3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reactivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NH2-HPTLC plate, reductive amination was found to be superior to the heating method reported earlier.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Higashi H,Hirabayashi Y,Ito M,Yamagata T,Matsumoto M,Ueda S,Kato S

doi

10.1093/oxfordjournals.jbchem.a122053

subject

Has Abstract

pub_date

1987-08-01 00:00:00

pages

291-6

issue

2

eissn

0021-924X

issn

1756-2651

journal_volume

102

pub_type

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