1. 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:
Chloroform Methanol
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°C 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°C, then process this second extract as in (4).
6. Rotovap (bath temperature not higher than 20-25°C) 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°C 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°C 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:
Di-isopropyl ether 1-butanol, reagent grade
Double distilled deionized H2O (ddH2O)
Saline solution: 0.1% NaCl or 0.3% NaCl
15 or 50 ml glass conical Corning Centrifuge tubes with teflon- lined caps (#8142)
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:
109 cells=1g tissue, so use 10 ml saline per g tissue.
Note: The details of the steps of the procedure below are critical and must be observed. Once step (1) is initiated, the procedure through step (7) should be carried out without delay.
1. Add organic (DIPE/1-butanol) phase to the total lipid extract; vortex, and sonicate until sample is well dispersed. An opalescent solution may result, but large particles must be dispersed before going to (2).
2. Add saline.
3. Perform a 2 min. combination of alternating vortexing and sonication.
4. Centrifuge for 10 min. at 2000 rpm (750xg).
5. Remove upper organic phase from lower aqueous phase (leave slight interface emulsion, when present, with the aqueous phase).
6. Add the original volume of fresh organic phase, repeat steps (3), (4), and (5) for the 2nd partitioning.
Note: Two partitionings for subsequent ganglioside autoradiographic studies, and up to three for tissue and plasma samples where highest qualitative purity is desired.
7. Prepare the final aqueous (ganglioside-containing) phase for STEP (3) below by removing traces of solvent with a stream of N2, freeze and lyophilize.
3. Sephadex G-50 gel filtration
Materials:
Sephadex G-50 (150) Eluent: double-distilled deionized water (ddH2O)
UV monitor and recorder, measure at 206 nm
Protocol:
Redissolve the sample in the appropriate volume of (ddH2O), briefly sonicate to ensure micelle formation and load the sometimes colored but always clear aqueous phase. Gangliosides are recovered in the void volume (peak 1) which is then lyophilized and redissolved in a small volume of C:M 1:1. The sample is centrifuged and the gangliosides are quantitatively recovered in the clear supernate. The precipitate contains residual protein coextracted in the total lipid extraction step.
References:
(1) Ladisch, S., and Gillard, B. (1985) A solvent partition method for micro-scale ganglioside purification. Analytical Biochemistry 146: 220-231.
(2) Ladisch, S., and Gillard, B. (1987) Isolation and purification of gangliosides from plasma. Methods in Enzymology 138: 300-306.
Typical column conditions: BED VOLUME FLOW RATE (ML/MIN) OPTIMAL (MAX) SAMPLE LOADING VOLUME TOTAL GANGLIOSIDE CONTENT OF LOADED SAMPLE GANGLIOSIDE RECOVERY 10-15 ML 0.25 0.3 (0.7) 5-20 80-100% 40 ML 0.6 0.5 (1.5) 20-50 100% 90 ML 1.2 1.5 (3.0) 70-5400 93-100%
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