Special Considerations for Glycolipids and Their Purification

David F. Smith1, Pedro A. Prieto2

1 The University of Georgia, Atlanta, Georgia, 2 Ross Laboratories, Columbus, Ohio
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.3
DOI:  10.1002/0471142727.mb1703s22
Online Posting Date:  May, 2001
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Abstract

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Extraction and Purification of Glycolipids from Cells or Tissues
  • Support Protocol 1: Preparation of Sep‐Pak C18 Cartridges
  • Basic Protocol 2: Preparation of Gangliosides
  • Alternate Protocol 1: Rapid Purification of Gangliosides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction and Purification of Glycolipids from Cells or Tissues

  Materials
  • Tissue sample or cell culture from one to several culture plates
  • Methanol (HPLC grade)
  • Chloroform (HPLC grade)
  • 4:8:3 (v/v/v) chloroform/methanol/water
  • 1:1 and 2:1 (v/v) chloroform/methanol
  • 100% ethanol
  • 0.1 M KCl
  • 1:1 (v/v) methanol/ 0.1 M KCl
  • 1:1 (v/v) methanol/H 2O
  • Sep‐Pak C 18 cartridge ( protocol 2support protocol)
Blender (Waring), tip sonicator (Branson), or Tissuemizer (Tekmar) equipped with a probe of appropriate size
  • Bath sonicator
  • Rotary evaporator, Speedvac (Savant), Vortex‐Evaporator (Labconco), or nitrogen dryer
1.5‐ml polypropylene microcentrifuge tubes resistant to chloroform or 12‐ml conical glass centrifuge tubes
  • Additional reagents and equipment for quantitation of protein and (unit 10.1), protein electrophoresis (units 10.2 10.5)

Support Protocol 1: Preparation of Sep‐Pak C18 Cartridges

  Additional Materials
  • 0.1 M ammonium acetate in 1:1 (v/v) methanol/water
  • Sep‐Pak C 18 cartridges (Waters)
  • 10‐ml glass syringe with locking hub (e.g., Becton Dickinson Luer‐Lok)

Basic Protocol 2: Preparation of Gangliosides

  Materials
  • Methanol
  • DEAE‐Sephadex equilibrated in 100% methanol, 50% slurry ( )
  • Purified Folch upper‐phase fraction (first protocol 1basic protocol)
  • 0.01, 0.2, and 0.5 M ammonium acetate in methanol
  • Glass wool

Alternate Protocol 1: Rapid Purification of Gangliosides

  Additional Materials
  • recipeDEAE‐Sephadex equilibrated in 60:30:8 methanol/chloroform/water ( reagents and solutions)
  • Total lipid extract (step of first protocol 1basic protocol)
  • 60:30:8 (v/v/v) methanol/chloroform/water
  • 60:30:8 (v/v/v) methanol/chloroform/0.8 M aqueous KCl
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Figures

Videos

Literature Cited

Literature Cited
   Clark, G.F., Cummings, R.D., Mattox, S., Gorbea, C., and Smith, D.F. 1991. Decreased biosynthesis of Forssman glycolipid after retinoic acid‐induced differentiation of mouse F9 teratocarcinoma cells: Lectin affinity chromatography of the glycolipid‐derived oligosaccharides. Carbohydr. Res. 213:155‐168.
   Doering, T.L., Masterson, W.J., Hart, G.W., and Englund, P.T. 1990. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J. Biol. Chem. 265:611‐614.
   Esselman, W.J., Laine, R.A., and Sweeley, C.C. 1972. Isolation and characterization of glycosphingolipids. Methods Enzymol. 28:140‐159.
   Finne, J. and Krusius, T. 1982. Preparation and fractionation of glycopeptides. Methods Enzymol. 83:269‐277.
   Folch, J., Lees, M.B., and Sloane Stanley, G.H. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226:497‐503.
   Hakomori, S. 1990.90. Bifunctional role of glycosphingolipids. J. Biol. Chem. 265:18713‐18716.
   Kannagi, K., Watanabe, K., and Hakomori, S. 1987. Isolation and purification of glycosphingolipids by high performance liquid chromatography. Methods Enzymol. 138:3‐12.
   Karlsson, K.‐A. 1989. Animal glycosphingolipids as membrane attachment sites for bacteria. Ann. Rev. Biochem. 58:309‐350.
   Laine, R.A. and Hsieh, T.C.‐Y. 1987. Inositol‐containing sphingolipids. Methods Enzymol. 138:186‐195.
   Ledeen, R.W. and Yu, R.K. 1982. Gangliosides: Structure, isolation and analysis. Methods Enzymol. 83:139‐191.
   McNeil, M., Chatterjee, D., Hunter, S.W., and Brennan, P.J. 1989. Mycobacterial glycolipids: Isolation, structures, antigenicity, and synthesis. Methods Enzymol. 179:215‐242.
   Macher, B.A. and Sweeley, C.C. 1978. Glycosphingolipids: Structure, biological source, and properties. Methods Enzymol. 50:236‐250.
   Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H., and Ginsburg, V. 1982. A monoclonal antibody‐defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto‐N‐fucopentaose II. J. Biol. Chem. 257:143‐14369.
   Makaaru, C.K., Damian, R.T., Smith, D.F., and Cummings, R.D. 1992. The human blood fluke Schistosoma mansoni synthesizes a novel type of glycosphingolipid. J. Biol. Chem. 267:2251‐2257.
   Momoi, T., Ando, S., and Nagai, Y. 1976. High resolution preparative chromatography system for gangliosides using DEAE‐Sephadex and the new porous silica, iatrobeads. Biochim. Biophys. Acta 441:488‐497.
   Smith, D.F., Larsen, R.D., Mattox, S., Lowe, J.B., and Cummings, R.D. 1990. Transfer and expression of murine UDPGal:β‐D‐Galα1,3Galactosyltransferase gene in transfected Chinese hamster ovary cells. J. Biol. Chem. 265:6225‐6234.
   Stults, C.L.M., Sweeley, C., and Macher, B.A. 1990. Glycosphingolipids: Structure, biological source, and properties. Methods Enzymol. 179:167‐214.
   Svennerholm, L. and Fredman, P. 1980. A procedure for the quantitative isolation of brain gangliosides. Biochim. Biophys. Acta 617:97‐109.
   Weigandt, H. 1985. Glycolipids. In New Comprehensive Biochemistry (A. Neuberger and L.L.M. van Deenen, eds.) Vol. 10. pp. 2‐4. Elsevier Science Publishing, New York.
   Williams, M.A. and McClure, R.H. 1980. The use of Sep‐Pak C18 Cartridge during the isolation of gangliosides. J.Neurochem. 35:266‐269.
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