The Isolation of Detergent-Resistant Lipid Rafts for Two-Dimensional Electrophoresis

  • Ki-Bum Kim
  • Jae-Seon Lee
  • Young-Gyu Ko
Part of the Methods in Molecular Biology™ book series (MIMB, volume 424)


Because lipid rafts are plasma membrane platforms mediating various cellular events such as in signal transduction, immunological response, pathogen invasion, and neurodegenerative diseases, protein identification in the rafts could provide important information to study their function. Here, we present an optimized method to isolate detergent-resistant lipid rafts that are subsequently analyzed by two-dimensional electrophoresis (2-DE). Lipid rafts were isolated based on their two distinct biochemical properties such as Triton X-100 insolubility and low density. To solubilize completely the proteins embedded in lipid rafts, sample lysis buffer (9 M urea, 2 M thiourea, 100 mM DTT, 2% CHAPS (w/v), 60 mM n-octyl -D-glucopyranoside, 2% IPG buffer) was applied to the isolated rafts. This method was found to be the most suitable choice for obtaining 2-DE profile of lipid raft proteome from various cells and tissues. We expect that this method could provide the way to dissect the function of raft-associated proteins and to gain a comprehensive insight upon various cellular events mediated through lipid rafts, the specialized domains in cell surface.

Key Words

detergent-resistant lipid rafts Two-dimensional electrophoresis 



This work was supported by grants awarded to YK from KOSEF (R01-2004-000-10765-0), Korea Research Foundation (KRF-2006-311-C00407), and Top Brand Project of Korea Basic Science Institute.


  1. 1.
    Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115, 377–388.CrossRefPubMedGoogle Scholar
  2. 2.
    Edidin, M. (2003) The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283.CrossRefPubMedGoogle Scholar
  3. 3.
    Lagerholm, B. C., Weinreb, G. E., Jacobson, K., and Thompson, N. L. (2005) Detecting microdomains in intact cell membranes. Annu. Rev. Phys. Chem. 56, 309–336.CrossRefPubMedGoogle Scholar
  4. 4.
    London, E. (2005) How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells. Biochim. Biophys. Acta. 1746, 203–220.CrossRefPubMedGoogle Scholar
  5. 5.
    Mukherjee, S., Maxfield, F.R. (2004) Membrane domains. Annu. Rev. Cell. Dev. Biol. 20, 839–866.CrossRefPubMedGoogle Scholar
  6. 6.
    Brown, D. A., Rose, J. K. (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544.CrossRefPubMedGoogle Scholar
  7. 7.
    Roper, K., Corbeil, D., Huttner, W. B. (2000) Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat. Cell Biol. 2, 582–592.CrossRefPubMedGoogle Scholar
  8. 8.
    Kim, K. B., Kim, S. I., Choo, H. J., and Ko, Y. G. (2004) Two dimensional electrophoretic analysis reveals that detergent resistant lipid rafts in physiological condition. Proteomics 4, 3527–3535.CrossRefPubMedGoogle Scholar
  9. 9.
    Anderson, R. G. (1998) The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225.CrossRefPubMedGoogle Scholar
  10. 10.
    Mineo, C., Gill, G. N. and Anderson, R. G. (1999) Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem. 274, 30636–30643.CrossRefPubMedGoogle Scholar
  11. 11.
    Simons, K., Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39.CrossRefPubMedGoogle Scholar
  12. 12.
    Manes, S., del Real, G., and Martinez-A, C. (2003) Pathogens: raft hijackers. Nat. Rev. Immunol. 3, 557–568.CrossRefPubMedGoogle Scholar
  13. 13.
    Gaus, K., Rodriguez, M., Ruberu, K. R., Gelissen, I., Sloane, T. M., Kritharides, L., Jessup, W. (2005) Domain-specific lipid distribution in macrophage plasma membranes. J. Lipid Res. 46, 1526–1538.CrossRefPubMedGoogle Scholar
  14. 14.
    Nabi, I. R., Le, P. U. (2003) Caveolae/raft-dependent endocytosis. J. Cell Biol. 161, 673–677.CrossRefPubMedGoogle Scholar
  15. 15.
    Bae, T. J., Kim, M. S., Kim, J. W., Kim, B. W., Choo, H. J., Lee, J. W., Kim, K. B., Lee, C. S., Kim, J. H., Chang, S. Y., Kang, C. Y., Lee, S. W., Ko, Y. G.. (2004) Lipid raft proteome reveals ATP synthase complex in the cell surface. Proteomics 4, 3536–3548.CrossRefPubMedGoogle Scholar
  16. 16.
    Foster, L. J., De Hoog, C. L., Mann, M. (2003) Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc. Natl. Acad. Sci. USA 100, 5813–5818.CrossRefPubMedGoogle Scholar
  17. 17.
    Calvo, M., Enrich, C. (2000) Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver. Electrophoresis 21, 3386–3395.CrossRefPubMedGoogle Scholar
  18. 18.
    Kim, K. B., Lee, J. W., Lee, C. S., Kim, B. W., Choo, H. J., Jung, S. Y., Chi, S. G., Youn, Y. S., Yoon, G.. S., and Ko, Y. G. (2006) Oxidation-reduction respiratory chains and ATP synthase complex are localized in the detergent-resistant lipid rafts. Proteomics 6, 2444–2453.CrossRefPubMedGoogle Scholar
  19. 19.
    Blagoev, B., Kratchmarova, I., Ong, S. E., Nielsen, M., Foster, L. J., and Mann, M. (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat. Biotechnol. 21, 31–318.CrossRefGoogle Scholar
  20. 20.
    Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., and Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999.CrossRefPubMedGoogle Scholar
  21. 21.
    Griffin, T. J., Tang, W., Smith, L. M. (1997) Genetic analysis by peptide nucleic acid affinity MALDI-TOF mass spectrometry. Nat. Biotechnol. 15, 1368–1372.CrossRefPubMedGoogle Scholar
  22. 22.
    Miranker, A., Robinson, C. V., Radford, S. E., Aplin, R. T., and Dobson, C. M. (1993) Detection of transient protein folding populations by mass spectrometry. Science 262, 896–900.CrossRefPubMedGoogle Scholar
  23. 23.
    Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A., and Simons, K. (2003) Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA 100, 5795–5800.CrossRefPubMedGoogle Scholar
  24. 24.
    Chamberlain, L. H. (2004) Detergents as tools for the purification and classification of lipid rafts. FEBS lett. 559, 1–5.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Ki-Bum Kim
    • 1
  • Jae-Seon Lee
    • 1
  • Young-Gyu Ko
    • 1
  1. 1.Korea UniversitySeoulKorea

Personalised recommendations