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

J-STAGE
  • HIGASHI Hideyoshi
    Department of Pathology, Research Institute for Microbial Diseases, Osaka University
  • HIRABAYASHI Yoshio
    Department of Biochemistry, Shizuoka College of Pharmacy
  • ITO Makoto
    Laboratory of Advanced Research, Mitsubishi-Kasei Institute of Life Sciences
  • YAMAGATA Tatsuya
    Laboratory of Advanced Research, Mitsubishi-Kasei Institute of Life Sciences
  • MATSUMOTO Makoto
    Department of Biochemistry, Shizuoka College of Pharmacy
  • UEDA Shigeharu
    Department of Pathology, Research Institute for Microbial Diseases, Osaka University
  • KATO Shiro
    Department of Pathology, Research Institute for Microbial Diseases, Osaka University

Abstract

A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccha-rides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4er, II3NeuGc-GgOse3e3Cer, 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 reac-tivity 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 NI-12-1-IPTLC plate, reductive amination was found to be superior to the heating method reported earlier.

Journal

  • J Biochem (Tokyo)

    J Biochem (Tokyo) 102 (2), 291-296, 1987

    The Japanese Biochemical Society

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