Nondestructive detection of gangliosides with lipophilic fluorochromes and their employment for preparative high-performance thin-layer chromatography.
A simple and effective procedure for the isolation and purification of gangliosides by preparative thin-layer chromatography is described. The method is based on nondestructive visualization of gangliosides on silica gel-precoated thin-layer chromatography plates by staining with uncharged lipophilic fluorochromes. Fluorescent dyes were added in low concentrations into the mobile phase (0.002%, w/v) without any interference of the ganglioside separation. After uv localization, the fluorescent zones were scraped off the plate and the silica gel was extracted with chloroform/methanol/water (30/60/8). In the following step fluorochromes were removed from gangliosides containing crude extracts by anion-exchange chromatography on DEAE-Sepharose. After desalting, impurities were removed by Iatrobeads chromatography. The method described offers an easy to handle and successful preparative thin-layer chromatography strategy to obtain pure gangliosides in microgram and miligram quantities.
Müthing
J
J
Institut für Zellkulturtechnik, Universität Bielefeld, Germany.
Heitmann
D
D
D000818:Animals; D001923:Brain Chemistry; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer / Q000379:methods*; D005069:Evaluation Studies as Topic; D005260:Female; D005456:Fluorescent Dyes; D005732:Gangliosides / Q000737:chemistry / Q000302:isolation & purification*; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data
D005456:Fluorescent Dyes; D005732:Gangliosides