23] Thin-layer chromatography of glycosphingolipids

Abstract

Glycosphingolipids are well suited for analysis by thin-layer chromatography (TLC), which is useful for monitoring purification, for qualitative and quantitative determination of expression in normal and pathological tissues, for partial structural analysis, and for detecting biological activities, including immunoreactivity and binding activity toward toxins, viruses, bacteria, and eukaryotic cells. Although quantitative TLC can be used to determine the concentration of resolved glycosphingolipid species, it is useful to estimate the total concentration of glycosphingolipid in a sample prior to or in association with TLC. Glycosphingolipid samples must first be freed of major contaminating lipids, proteins, and low molecular weight contaminants. The most widely used TLC developing solvents for glycosphingolipids are mixtures of chloroform, methanol, and water (or aqueous salts), because they form a single phase at a range of hydrophobicities well suited for glycosphingolipid resolution on silica gel TLC plates. Reversible stains are useful for preparative TLC. To improve recovery, the plate should not be allowed to dry thoroughly at any time after running. The simplest but least sensitive reversible staining method is to spray the TLC plate heavily with distilled water.