Procedure for isolation of gangliosides in high yield and purity: simultaneous isolation of neutral glycosphingolipids.
While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002845:Chromatography / Q000379:methods; D005732:Gangliosides / Q000302:isolation & purification*; D006028:Glycosphingolipids / Q000302:isolation & purification*; D009186:Myelin Sheath / Q000032:analysis; D011817:Rabbits; D014867:Water
D005732:Gangliosides; D006028:Glycosphingolipids; D014867:Water