Summary
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.
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References
Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115, 377–388.
Edidin, M. (2003) The state of lipid rafts: from model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283.
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.
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.
Mukherjee, S., Maxfield, F.R. (2004) Membrane domains. Annu. Rev. Cell. Dev. Biol. 20, 839–866.
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.
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.
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.
Anderson, R. G. (1998) The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225.
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.
Simons, K., Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39.
Manes, S., del Real, G., and Martinez-A, C. (2003) Pathogens: raft hijackers. Nat. Rev. Immunol. 3, 557–568.
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.
Nabi, I. R., Le, P. U. (2003) Caveolae/raft-dependent endocytosis. J. Cell Biol. 161, 673–677.
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.
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.
Calvo, M., Enrich, C. (2000) Biochemical analysis of a caveolae-enriched plasma membrane fraction from rat liver. Electrophoresis 21, 3386–3395.
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.
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.
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.
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.
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.
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.
Chamberlain, L. H. (2004) Detergents as tools for the purification and classification of lipid rafts. FEBS lett. 559, 1–5.
Acknowledgments
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.
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Kim, KB., Lee, JS., Ko, YG. (2008). The Isolation of Detergent-Resistant Lipid Rafts for Two-Dimensional Electrophoresis. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_32
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DOI: https://doi.org/10.1007/978-1-60327-064-9_32
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