A Method for Micro-Scale Isolation and Purification of Gangliosides

Stephan Ladisch~Director, Center for Cancer and Transplant Biology, Children's National Medical Center, Washington, D.C. 22010

This solvent partition method permits the isolation of gangliosides from small samples and from samples in which the ganglioside concentration is low, especially relative to the concentration of potentially contaminating proteins and other large molecular weight species. The method consists of three steps: (1) preparation of a dry total lipid extract, (2) partition of the total lipid extract in di-isopropyl ether/1-butanol/aqueous NaCl, and (3) Sephadex G-50 gel exclusion chromatography. The final total ganglioside preparation is lyophilized and stored in the dry state.

OVERALL STEPS

1. Total lipid extract of lyophilized cells, plasma, or tissue.

2. Partition of the dried total lipid extract between DIPE/1-butanol/ saline aqueous and organic phases.

3. Removal of low molecular weight contaminants from the ganglioside-containing aqueous phase by Sephadex G-50 gel filtration.

1. Total Lipid Extraction

Materials:

Protocol:

1. Lyophilize sample (cells, plasma, homogenized tissue) in a round bottom glass tube or flask of appropriate size (i.e. 30 x tissue or plasma volume).

2. Pulverize lyophilized samples to a fine powder with a glass rod. Add chloroform:methanol (C:M) 1:1. v/v (20 vol/gm wet wt tissue or cells, 10 vol/ml plasma or serum). Disperse solids with sonication in bath sonicator, blanket with N₂ and cap tightly. (For wet extraction, first add 10 vol. methanol with stirring, and then 10 vol. chloroform).

3. FIRST EXTRACTION

Extract for 18 hr. at 4癈 with magnetic stirring.

4. Centrifuge tubes at 2000 rpm (750 x g for 10 min.). carefully decant the completely clear supernate, without transferring any particulate matter, and process the supernate per (6) below.

5. SECOND EXTRACTION

Reextract the total solid material with an additional original volume of fresh C:M 1:1; use the fresh solvent to first rinse the centrifuge tubes used in (4), and then combine these rinses in the original extraction flask. Extract for 4 hr. with stirring, under N₂ at 4癈, then process this second extract as in (4).

6. Rotovap (bath temperature not higher than 20-25癈) the combined supernatants from (4) and (5) to reduce their volume to 1/4 of the combined total volume. At this point cool overnight to -25癈 to precipitate protein. Recentrifuge as above, and recover the clear supernate. Quanti-tatively transfer this concentrated total lipid extract to the centrifuge tube (#8142) in which the DIPE/butanol partition will be performed. Save the pellets until after the G-50 stage, in case reprocessing is necessary. Evap-orate the supernate to dryness with a stream of N₂, and remove remaining traces of solvent with lyophilization.

Notes: Over 90% of total gangliosides are removed by the first extraction, and the remaining 10% in the second extract is not qualitatively different from that in the first extract; the precipitate removed by cooling to -20癈 is free of gangliosides.

The volume of C:M used has not been found to be critical within a reasonable range. We have used up to 10 ml/10⁸ cells (i.e. a higher volume) with no effect on recovery compared to the usual 10-20 volumes.

2. DIPE/1-butanol/saline partition

Materials:

Protocol:

Phases:

Organic: 2 volumes DIPE/1-butanol, 60:40 v/v

Aqueous: 1 volume saline solution

Aqueous phases are as follows:

Cell pellets: for cells, use 0.l% NaCl:

10 ml saline per total lipid extract of 109 cells (minimum vol of 1 ml).

Plasma: for plasma, use ddH2O:

2.0 ml ddH₂O per totaI lipid extract of 1 ml plasma, minimum aqueous vol of 1 ml.

Radio-labelled gangliosides of cells: 1.0 ml minimum vol of saline (use with as little as 10⁶ Cells).

Tissue: for tissue, use 0.3% NaCl: