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

Hideyoshi Higashi, Yoshio Hirabayashi, Makoto Ito, Tatsuya Yamagata, Makoto Matsumoto, Shigeharu Ueda, Shiro Kato

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14 Citations (Scopus)

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, 14278-14278) and sialyllactose were chromatographed on polyamide 11 TLC or NH2HPTLC 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 NH2HPTLC 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.

Original languageEnglish
Pages (from-to)291-296
Number of pages6
JournalJournal of biochemistry
Volume102
Issue number2
DOIs
Publication statusPublished - Aug 1987

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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