Glycosphingolipids with chloroform/methanol, extraction

For preparation of CMHs, a lipid extract from fungal cells is obtained by successive extractions with chloroform/methanol (2 1 and 1 2 v/v). These extracts are usually combined and dried, yielding a crude lipid mixture. The crude extract is subsequently partitioned according to Folch et al. [27], in which the lower phase containing neutral glycosphingolipids is taken for further analysis. 

The neutral lipid fraction from the DEAE-Sephadex A-50 column was combined with the lower phase obtained after Folch partition of the total lipid extract and the combined lipids dried. To the same flask, 10Q ml of 0.6 M NaOH in methanol was added. The mixture was incubated at 37°C for 5 hours. Five volumes of acetone were then added and stored overnight at 4°C. The precipitate was collected by centrifugation at 4°C and dissolved in C M (4 1, v/v). After application to the column (2.0 x 25 cm), the column was washed with chloroform. Neutral glycolipids were then eluted with tetrahydrofuran H2O (10 1). Fractions containing neutral glycosphingolipids were pooled and their glycolipid content examined by thin-layer chromatography. 

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