Abstract
A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of125I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of125I-LFA is not inhibited by 106-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 μl of serum were sufficient for the detection of major gangliosides.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- LFA:
-
Limax flavus agglutinin
- ELLA:
-
Enzyme Linked Lectin Assay
- PIM:
-
Poly(isobutyl methacrylate)
- PVP:
-
Polyvinylpyrrolidone mol.wt. 40,000
- PBS:
-
Phosphate buffered saline
- BSA:
-
Bovine serum albumin
References
Svennerholm L (1957) Biochim Biophys Acta 24:604–11.
Aminoff D (1961) Biochem J 81:384–92.
Magnani JL, Smith DF, Ginsburg V (1980) Anal Biochem 109:399–402.
Magnani JL, Brockhaus M, Smith DF, Ginsburg V (1982) Methods Enzymol 83:235–41.
Saito M, Kasai N, Yu RK (1985) Anal Biochem 148:54–58.
Magnani JL, Brockhaus M, Smith DF, Ginsburg V, Blaszczyk M, Mitchell KF, Steplewski Z, Koprowski H (1981) Science 212:55–56.
Brockhaus M, Magnani JL, Blaszczyk M, Steplewski Z, Koprowski H, Karlsson KA Larson G, Ginsburg V (1981) J Biol Chem 256:13223–25.
Smith DF (1983) Biochem Biophys Res Commun 115:360–67.
Hansson GC, Karlsson KA, Larson G, Strömberg N, Thurin J (1985) Anal Biochem 146:158–63.
Swank-Hill P, Needham LK, Schnaar RL (1987) Anal Biochem 163:23–25.
Molin K, Fredman P, Svennerholm L (1986) FEBS Lett 205:51–52.
McCoy JP, Varani J, Goldstein IJ (1983) Anal Biochem 130:437–44.
Holmgren J, Elwing H, Fredman P, Svennerholm, L (1980) Eur J Biochem 106:301–79.
Goldstein IJ, Poretz RD (1986) in The Lectins, eds. Liener IE, Sharon N, Goldstein IJ, Academic Press, Orlando, p 209–14.
Monsigny M, Roche AC, Sene C, Maget-Dona R, Delmontte FM (1980) Eur J Biochem 104:147–53.
Yen SE, Mansfield JM, Wallace JH (1980) Int Arch Allergy Appl Immunol 61:32–39.
Mohan S, Dorai DT, Srimal S, Buchhawat BK (1982) Biochem J 203:253–62.
Miller RL (1982) J Invertebr Pathol 39:210–14.
Miller RL, Collawn JF, Fish WW (1982) J Biol Chem 257:7574–80.
Hunter WM (1967) in Handbook of Experimental Immunology, ed. Weir DK, Davis, Philadelphia, p 608–42.
Svennerholm L, Fredman P (1980) Biochim Biophys Acta 617:97–109.
Colman PG, Nayak RC, Campbell IL, Eisenbarth GS (1988) Diabetes 37:645–52.
Schauer R (1978) Methods Enzymol 50:64–89.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265–75.
Wenger DA, Wardall S (1973) Neurochem 20:607–12.
Tettamanti G, Bonali F, Marchesini S, Zambotti V (1973) Biochim Biophys Acta 296:160–70.
Tomasi M, Roda C, Ausiello C, D'Agnolo G, Venerando B, Ghidoni R, Sonnini S, Tettamanti G (1980) Eur J Biochem 111:315–24.
Brockhaus M (1985) in Immunological Methods Vol III, eds. Lefkovits I, Pernis B, Academic Press, Orlando, p 133–45.
Tettamanti G, Masserini M (1987) in Biomembrane and Receptor Mechanisms, Fidia Research Series Vol 7, eds. Bertoli E, Chapman D, Cambria A, Scapagnini U, Liviana Press, Padova, p 235.
Suzuki K (1965) J Neurochem 12:629–38.
Kracun I, Rösner H, Cosovic C, Stavljenic A (1984) J Neurochem 43:979–89.
Urban PF, Harth S, Freysz L, Dreyfus H (1984) Adv Exp Med Biol 174:104–10.
Tai T, Kawashima J, Tada N, Ikegami S (1988) Biochim Biophys Acta 958:134–38.
Kundu SK, Diego I, Osovitz S, Marcus DM (1985) Arch Biochem Biophys 238:388–400.
Ladisch S, Gillard B (1987) Methods Enzymol 138:300–6.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kielczyski, W., Harrison, L.C. Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms. Glycoconjugate J 7, 75–84 (1990). https://doi.org/10.1007/BF01050404
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01050404