Published: March 12th, 2021
Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.
Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.
Gangliosides are defined as glycosphingolipids bearing one or more sialic acid residues1. They are expressed primarily at the cell surface with their hydrophobic ceramide lipid moiety embedded in the outer leaflet of the plasma membrane and their hydrophilic glycans extending into the extracellular space2. Although distributed widely in vertebrate cells and tissues, they are particularly abundant in the vertebrate brain3, where they were first discovered and named4.
The structures of ganglioside glycans vary and are the basis for their nomenclatu....
Tissue collection was performed under conditions authorized by the Johns Hopkins Animal Care and Use Committee.
1. Small scale ganglioside extraction and partial purification
CAUTION: Use appropriate ventilation when working with volatile and toxic solvents. Avoid plastic throughout; solvents will extract chemical components from many plastics that interfere with subsequent analyses. Polytetrafluoroethylene (PTFE) is an exception; PTFE-lined closures should be used to.......
The methods described in section 1 (small scale) provide gangliosides at sufficient quantity and purity for qualitative and quantitative determination of major brain gangliosides. Recovery from mouse brain is ~ 1 µmol ganglioside per g brain wet weight (1 nmol/µL) when prepared as described. TLC resolution of 1 µL (1 nmol) using section 3 provides ample material for resorcinol detection and resolves all of the major brain gangliosides as shown for wild type and genetically modified mice in
The methods for small and large scale ganglioside extraction and isolation reported here are not unique - there are many different solvent extraction and purification approaches that provide excellent results12. The methods reported here for small scale purification from brain, from Fredman and Svennerholm13, were shown to optimize recovery and have proven to be robust and straightforward over many years in our laboratory. Isolation and purification suitable for TLC and MS .......
This work was supported by National Institutes of Health (NIH) Common Fund for Glycoscience grant U01CA241953. MJP was supported by the Chemistry-Biology Interface Program at Johns Hopkins (T32GM080189).....
|Bovine brain, stripped
|GM1, 1061; GD1a, 1062; GD1b, 1501; GT1b, 1063
|Glass bottle with PTFE-lined cap
|Glass centrifuge bottle
|Glass culture tubes, 16 x 125 mm
|for collecting HPLC fractions
|Glass separatory funnel (2 L)
|Injection syringe - Hamilton 1750 gastight 500 µl
|Choose appropriate volume option
|Reprosil 100 NH2 10µm 5x4mm guard columns
|Reprospher 100 NH2, 5 μm, 250 mm x 20 mm HPLC column
|other sizes available
|Sample loop for Model 7725 Injector (5 ml)
|Sep-Pak tC18 Cartidges Vac 35 cc (10 g)
|Sep-Pak tC18 Plus Short Cartridge, 400 mg
|Spotting syringe - Hamilton 701N 10 µl
|Thick-walled 13-mm diameter test tubes with PFTE lined caps
|Threaded 2-ml vials with PFTE lined caps
|For ganglioside storage
|TLC plates, HPTLC Silica gel 60 F254 Multiformat
|Fluorescence impregnation (F254) stabilizes the sorbent surface
|TLC reagent sprayer
|TLC running chamber for 10 x 10 cm plates
|Waring 1-Liter Stainless Steal Explosion Resistant Blender
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