Immunostaining on thin-layer chromatograms of oligosaccharides released from gangliosides by endoglycoceramidase.
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.
Higashi
H
H
Department of Pathology, Osaka University.
Hirabayashi
Y
Y
Ito
M
M
Yamagata
T
T
Matsumoto
M
M
Ueda
S
S
Kato
S
S
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer / Q000379:methods; D004912:Erythrocytes / Q000032:analysis; D005732:Gangliosides / Q000097:blood; D006026:Glycoside Hydrolases; D007118:Immunoassay / Q000379:methods; D009844:Oligosaccharides / Q000032:analysis* / Q000276:immunology; D013552:Swine
D005732:Gangliosides; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase