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Bonali, S. Marchesini, and V. Zambotti, Biochim. Biophys. Acta 296, 160 (1973). 2~ y. Suzuki and K. Suzuki, J. LipidRes. 13, 687 (1972). [3] AFFINITY CHROMATOGRAPHY OF GLYCOLIPIDS 37 Procedures. Lipid extracts were prepared from lyophilized membranes as described previously? 5) and then separated, when necessary, into neutral and acidic lipid fractions on DEAE-Sepharose according to the method of Ledeen and Yu. 29 Working solutions of lipid extracts or glycolipid fractions were prepared in chloroform/methanol (2/1) so that 1 ml of solution was equivalent to 100 mg of membrane protein.
Biol. Chem. 255, 6633 (1950). t4 M. Fukuda, M. Lauffenburger, H. Sasaki, M. E. Rogers, and A. Dell, J. Biol. Chem. 262, 11952 (1987). 22 ANALYTICALMETHODS [2] 16 ml). 5 ml are collected. 4, in order to elute highly charged materials. Ion-exchange HPLC is carried out as follows. 0. 0 (40 ml each). 0 ml/min, and the eluate is collected every 30 sec. 3-ml fractions. In fact, both QAE-Sephadex and TSK-DEAE column chromatographies allow fractionation of oligosaccharides with the same charge but different core saccharides under suitable conditions (Fig.
261, 14278(1986). 9 S. Li, R. DeGasperi, J. E. Muldrey,and Y. Li, Biochem. Biophys. Res. Commun. 141, 346 (1986). 1oR. D. Cummings and S. Kornfeld, J. Biol. Chem. 257, 11235(1982). " B. V. Torres and D. F. Smith, Anal. Biochem. 170, 209 (1988). METHODS IN ENZYMOLOGY. VOL. 179 Copyright© 1989by AcademicPress,Inc. All rightsof reproductionin any formreserved. [3] AFFINITY CHROMATOGRAPHY OF GLYCOLIPIDS 31 TABLE I STRUCTURES OF GLYCOLIPIDS Name GM2 Asialo-G~2 (gangliotriosyiceramide) Forssman glycolipid Globoside A type 1 hexaosylceramide Structure GalNAc(fll - 4)Gal(fl1-4)Glc-Cer 3 NeuAc(ct2) GalNac(fll - 4)Gal(fll - 4)Glc-Cer GalNAc(ot I - 3)GaINAc(fll - 3)Gal(a1-4)Gal(fll - 4)Glc-Cer GalNAc(fll - 3)Gal(a1-4)Gal(fll - 4)Glc-Cer G a l N A c ( a 1 - 3)Gal(fll - 3)GlcNAc(fll - 3 )Gal(fll - 4)Glc-Cer 2 Fuc(od) Dolichos biflorus (DB) lectin was provided by Dr.