Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase.
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A method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. <i>N</i>-Glycolylneuraminic acid (NeuGc)-containing oligosaccha-rides derived from II<sup>3</sup>NeuGc-LacCer, IV<sup>3</sup>NeuGc-nLcOse<sub>4</sub>er, II<sup>3</sup>NeuGc-GgOse3e<sub>3</sub>Cer, and II<sup>3</sup>(NeuGc)<sub>2</sub>-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) <i>J. Biol. Chem.</i> 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH<sub>2</sub>-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH<sub>3</sub>-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II<sub>3</sub>NeuGc-nLcOse<sub>4</sub> showed the highest reac-tivity with the antibody, followed by II<sub>3</sub>NeuGc-GgOse<sub>3</sub>. 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 NH<sub>2</sub>-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.
- J Biochem (Tokyo)
J Biochem (Tokyo) 102(2), 291-296, 1987
The Japanese Biochemical Society