Solubilization of Lymphocytes

Allan M. Weissman1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.1A
DOI:  10.1002/0471142735.im0801as57
Online Posting Date:  November, 2003
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Abstract

Purification and study of transmembrane proteins require isolation of these structures from their lipid environment. This isolation is carried out through the use of detergents. In this unit, several approaches to solubilization of membrane proteins are presented. Solubilization of whole lymphocytes, using conditions aimed at minimizing the disruption of protein‐protein interactions, is described with an optional step that may be useful when the disruption of protein interactions is desired as part of a purification protocol. In some situations, it may be desirable to purify membranes prior to their solubilization or to determine the physical relationship between proteins, which can be accomplished by a cross‐linking.

     
 
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Table of Contents

  • Solubilization of Cellular Proteins
  • Basic Protocol 1: Nonionic Detergent Solubilization of Lymphocytes
  • Basic Protocol 2: Preparation of Membranes by Dounce Homogenization
  • Alternate Protocol 1: Preparation of Membranes by Nitrogen Cavitation
  • Support Protocol 1: Cross‐Linking Membrane Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Nonionic Detergent Solubilization of Lymphocytes

  Materials
  • Cultured cells
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • Triton X‐100 lysis buffer with protease inhibitors (see recipe; store at 4°C)
  • 10% sodium deoxycholate (Na‐DOC), room temperature (optional; Sigma)
  • 10% SDS, room temperature (optional; Bio‐Rad)
  • Additional reagents and equipment for harvesting and counting cells ( appendix 3A3)
NOTE: Carry out all steps in this protocol at 4°C or on ice.

Basic Protocol 2: Preparation of Membranes by Dounce Homogenization

  Materials
  • Cultured cells
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • Dounce buffer with protease inhibitors (see recipe), ice‐cold
  • Tonicity restoration buffer with protease inhibitors (see recipe)
  • 0.5 M EDTA, pH 7.6
  • Triton X‐100 lysis buffer with protease inhibitors (see recipe)
  • Dounce homogenizers with type B tight‐fitting pestle Kontes), chilled to 4°C
  • Phase‐contrast microscope
  • 10‐ml polycarbonate ultracentrifuge tubes
  • Beckman Ti‐70.1 or equivalent horizontal or fixed‐angle ultracentrifuge rotor
  • Potter‐Elvehjem grinder (optional; Kontes) Triton X‐100 lysis buffer with protease inhibitors
NOTE: Carry out all steps in this protocol at 4°C or on ice.

Alternate Protocol 1: Preparation of Membranes by Nitrogen Cavitation

  Additional Materials
  • Nitrogen cavitation device (Parr Instrument) with nitrogen gas tank
NOTE: Carry out all steps in this protocol at 4°C or on ice.

Support Protocol 1: Cross‐Linking Membrane Proteins

  Additional Materials
  • Cross‐linking buffer (see recipe)
  • Disuccinimidyl suberate (DSS; Pierce) or dithiobis(succinimidyl propionate) (DSP; Pierce)
  • Dimethylsulfoxide (DMSO; reagent‐grade, Fisher)
  • TE buffer, pH 7.4 ( appendix 2A)
NOTE: Carry out all steps in this protocol at 4°C or on ice.
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Figures

Videos

Literature Cited

   Evans, W.H. 1978. Preparation and Characterization of Mammalian Plasma Membranes. (T.S. Work and E. Work, eds.) Elsevier, New York.
   Fletcher, S. and Packer, L. (eds.) 1974. Biomembranes, Part A. Methods Enzymol. 31:1‐478.
   Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734‐749.
   Hjelmeland, J.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. Methods Enzymol. 104:305‐318.
   Ji, H.T. 1983. Bifunctional reagents. Methods Enzymol. 91:580‐609.
   Kinet, J‐P., Quarto, R., Perez‐Montfort, R., and Metzger, H. 1985. Noncovalently and covalently bound lipid on the receptor for immunoglobulin E. Biochemistry 24:7342‐7348.
   Oettgen, H.C., Pettey, C.L., Maloy, W.L., and Terhorst, C. 1986. A T3‐like protein complex associated with the antigen receptor on murine T cells. Nature (Lond.) 320:272‐275.
   Rivnay, B. and Metzger, H. 1982. Reconstitution of the receptor for immunoglobulin E into liposomes: Conditions for incorporation of the receptor into vesicles. J. Biol. Chem. 257:12800‐12808.
   Rivnay, B., Wank, S.A., Poy, G., and Metzger, H. 1982. Phospholipids stabilize the interaction between the α and β subunits of the solubilized receptor for immunoglobulin E. Biochemistry 21:6922‐6927.
   Samelson, L.E., Harford, J., and Klausner, R.D. 1985. Identification of the components of the murine T cell antigen receptor complex. Cell 43:223‐231.
   Sharon, M., Klausner, R.D., Cullen, B.R., Chizzonite, R., and Leonard, W.J. 1986. Novel interleukin‐2 receptor subunit detected by cross‐linking under high affinity conditions. Science 234:859‐863.
   van Renswoude, J. and Kempf, C. 1984. Purification of integral membrane proteins. Methods Enzymol. 104:329‐339.
   Weissman, A.M., Samelson, L.E., and Klausner, R.D. 1986. A new subunit of the T cell antigen receptor. Nature (Lond.) 324:480‐482.
   Weissman, A.M., Baniyash, M., Hou, D., Samelson, L.E., Burgess, W.H., and Klausner, R.D. 1988. Molecular cloning of the zeta chain of the T cell antigen receptor. Science 239:1018‐1021.
   Welling, G.W., van der Zee, R., and Welling‐Wester, S. 1987. Column liquid chromatography of integral membrane proteins. Chromatography. 418:223‐243.
Key References
   Deutscher, M.P. (ed.) 1990. Guide to protein purification. Methods Enzymol. 182:203‐282.
  Discusses isolation of subcellular organelles (pp. 203‐225), preparation of membrane fractions (pp. 225‐235), and details of detergent solubilization (pp. 239‐282).
   Helenius et al., 1979. See above.
  Detailed discussion of properties and use of detergents to solubilize proteins.
   Hjelmeland et al., 1984. See above.
  Details on solubilizing membrane proteins.
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