Column chromatography gangliosides

Buffered Tetrahydrofuran. In 1973, Tettamanti et al. [19) described an improved procedure for the extraction, separation and purification of brain gangliosides. In this method, the brain tissue was subjected to homogenization and extraction with buffered [potassium phosphate buffer, pH 6.8) tetrahydrofuran. Following centrifugation, diethyl ether was added and the mixture separated into organic and aqueous phase. The gangliosides, recovered exclusively in the aqueous phase, were then freed of residual phospholipids and other minor contaminants [i.e. peptides)by column chromatography on silica gel.This procedure, as shown by the authors,was superior to the commonly used chloroform/methanol... 

Column Chromatography. Sepharose beads containing covalently linked gangliosides (0.2 ml packed volume) were placed into a pasteur pipette containing a small amount of glass wool. Columns were washed with HEM containing 50 ug/ml bovine serum albumin (3 ml). Interferon solutions in MEM-albumin (1 ml) were placed on the columns, which were eluted with MEM-albumin at a flow rate of no more than one drop per minute. Fractions of 1 ml were collected and interferon titers determined in each fraction after serial two-fold dilution. Columns onto which mouse fibroblast interferon had been loaded, were eluted with MEM-albumin first, then with 0.07 M N-acetylneuraminyl lactose at pH 2. 

Further characterization of Thy-1 active glycolipids was accomplished by fractionating brain gangliosides into mono-, di- and trisialo-gangliosides by DEAE column chromatography according to the procedure described by Nagai (14). These fractions eluted from the column were tested for Thy-1 activity (Table III). 

Monosialyltetrahexosylceramide (Ghi) and di-sialyltetrahexosylceramide (Gdi ) were isolated from bovine brain total gangliosides by silicic acid column chromatography (Svennerholm et a/., 1972). 

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

The substrate, fucosyl-GMj, was separated from its product, GMj, by chromatography on a LiChrosorb-NH2 column (4 mm X 250 mm, 5 /urn). The mobile phase was a 25 75 mixture of 5 mM potassium phosphate buffer (pH 5.5) and acetonitrile. Similarly, fucosyl-GDib was separated from GDib. except the concentration of phosphate buffer was 20 mM Gangliosides were detected by their absorbance at 195 nm. 

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