Gangliosides are expressed on all vertebrate cell surfaces and are especially abundant on nerve cells. They regulate cell signaling and cell-cell recognition. This protocol introduces ganglioside isolation and analysis.
The techniques were selected to maximize ganglioside yields at large and small scales while streamlining purification. We also describe methods to relatively quickly perform qualitative and semiquantitative ganglioside analyses. Study on lipids, especially polar lipids like gangliosides, requires different tools and techniques than working with proteins or nucleic acids.
This video familiarizes you with how to work with these molecules. Because solvents such as chloroform and tetrahydrofuran dissolve common plastics, we use glass bottles and vials with Teflon-lined caps, glass pipettes, glass, stainless-steel microsyringes, and Teflon glass homogenizers throughout these procedures. Failure to do so results in plastic polymers in the products that can interfere with analyses.
To begin, add 4.1 milliliters of water per gram of tissue wet weight, and homogenize it with 10 strokes. Add 13 milliliters of methanol per gram of tissue. Transfer it to an ambient temperature of 22 degrees Celsius, and mix.
Transfer the tissue to a thick-walled, glass, screw-capped tube with a PTFE-lined screw cap, and mix thoroughly. Add 6.5 milliliters of chloroform per gram of tissue, cap it, and mix thoroughly. Centrifuge at 450 times g for 15 minutes.
Then transfer the clear supernatant to a fresh, screw-capped tube, and measure the volume as recovered extract volume. For partitioning, add 0.173 times the recovered extract volume of water to the clear supernatant. Then cap it.
Vortex vigorously. Centrifuge at 450 times g for 15 minutes. Transfer the upper phase, which contains the gangliosides, into a fresh, glass tube with a PTFE-lined screw cap.
To perform reverse-phase cartridge chromatography, use a five-milliliter glass syringe to wash a tC18 solid-phase extraction cartridge with three milliliters of methanol. Then add three milliliters of chloroform-methanol-water at a ratio of two to 43 to 55. Load the upper phase from the previous step onto the tC18 cartridge through the same glass syringe.
Collect the flow-through, and reload it onto the column to optimize adsorption. Wash the cartridge with three milliliters of chloroform-methanol-water and then with three milliliters of methanol-water. Elute the gangliosides with three milliliters of methanol into a fresh, screw-capped tube.
Evaporate to dryness under a gentle stream of nitrogen at a temperature less than or equal to 45 degrees Celsius. Dissolve in methanol at one milliliter per gram of original tissue wet weight. Place 100 grams of brain gray matter in a blender, and add one milliliter per gram brain wet weight of chilled, 10-millimolar potassium phosphate buffer of pH 6.8.
Then homogenize on low for 20 seconds. Add eight milliliters of tetrahydrofuran per gram brain wet weight, and homogenize on low for 10 seconds. Decant into glass centrifuge bottles, and centrifuge at 5, 000 times g for 15 minutes.
Collect the supernatant, measure its volume, and then transfer it to a glass separatory funnel. Add 0.3 milliliters of ethyl ether per milliliter of the supernatant. Shake vigorously.
Then allow it to sit undisturbed for 30 minutes, during which two phases, an upper ether phase and a lower aqueous phase, separate. Collect the lower phase, which contains the gangliosides, into a glass bottle with a PTFE-lined cap. To the upper ether phase remaining in the separatory funnel, add 0.1 milliliters of water per milliliter of original supernatant from the previous step.
Shake vigorously. Allow phases to separate. Then collect the lower aqueous phase, and combine it with the previously collected lower phase.
Evaporate the combined lower phases to a dry powder, and weigh it. To perform reverse-phase chromatography, prewash a large-scale tC18 solid-phase extraction cartridge by passing 50 milliliters of solvents through the column using vacuum or pressure for less than one minute per wash. Load the upper phase from the post-saponification phase separation onto the column by vacuum or pressure.
Then collect the flow-through, reload it, and collect the flow-through again for subsequent analysis. Wash the column with 30 milliliters each of chloroform-methanol-water, followed by methanol-water. Then elute the gangliosides with 50 milliliters of methanol and then again with 10 milliliters of methanol.
Collect each wash and elution separately. Use thin-layer chromatography, or TLC, to confirm that gangliosides are absent from the flow-through and washes and eluted in the first elution of methanol. Evaporate the eluted gangliosides to a dry powder, and weigh them.
To perform TLC, pour running solvent into a 10-by-10 centimeter, glass TLC chamber with a stainless-steel cover so that the solvent depth is 0.5 centimeters. Cover the chamber, and allow it to equilibrate for about 10 minutes. Wash a 10-microliter Hamilton syringe with a beveled needle using methanol.
Draw one microliter of methanol into a glass syringe to fill the needle dead volume and then one microliter of sample or standard. Spot the sample evenly onto the five-millimeter, pre-marked lines until less than one microliter of methanol solvent remains in the syringe. Allow the plate to dry at 22 degrees Celsius after all samples are spotted.
Place the spotted and dried plate onto the pre-equilibrated TLC chamber with the bottom edge immersed in the running solvent. Allow the running solvent to advance up the plate by capillary action until the solvent front reaches within one centimeter of the top of the plate. Remove and mark the solvent front at the edge of the plate with a pencil.
Allow the solvents to evaporate completely either undisturbed or under mild air flow. In a chemical fume hood, place the TLC plate with the resolved gangliosides origin end upside down in a cut-away cardboard box to protect the walls of the hood from acid spray. Place resorcinol spray reagent in a glass TLC sprayer.
Attach it to a source of pressurized nitrogen, and lightly spray the plate diagonally in the vertical and horizontal directions. Immediately cover the plate with a clean, dry, glass cover plate of the same dimensions, and secure the cover plate in place with binder clips. Heat the covered plate at 125 degrees Celsius for 20 minutes.
Gangliosides will appear dark purple against a white background. To perform lipid staining, dip the TLC plate with the resolved gangliosides origin side down into a beaker containing the anisaldehyde stain. Submerge to the running front for just over two seconds.
Then allow it to drain. Heat the TLC plate on a hot plate at a low temperature to develop. This protocol provides gangliosides at sufficient quantity and purity for qualitative and quantitative determination.
TLC resolution of one microliter provides ample material for resorcinol detection and resolves all of the major brain gangliosides for wild-type and genetically modified mice. Although the prepared mixed gangliosides are not free of other major lipids, they are of sufficient purity for mass spectrometric determination either as native purified gangliosides in negative mode or after pre-methylation in positive mode. Large-scale purification includes extraction, saponification, and HPLC resolution to provide purified major brain gangliosides suitable for biological experiments and for further chemical and enzymatic modifications.
An exemplary HPLC profile and subsequent TLC analysis are shown. Alkali treatment, or saponification, is necessary to hydrolyze and remove contaminating phospholipids but will also hydrolyze natural modifications of gangliosides such as O-acetylated sialic acids, which may be important in some contexts. Accurate solvent ratios for extraction, solvent partitioning, and TLC resolution are critical to enhance recovery, purity, and analyses.
From cancer to metabolism to brain function, gangliosides are physiological modifiers and therapeutic targets. Ganglioside isolation, purification, and analyses are cornerstones for biomedical discovery and therapeutic development.
