Abstract
Isolation of detergent-resistant membranes (DRMs; also known as detergent-insoluble glycolipid-enriched membranes [DIGs] or glycolipid-enriched membranes [GEMs]) that are enriched in proteins and lipids with a high affinity for rafts is one of the simplest and most widely used methods for studying rafts. However, it is essential to understand the limitations as well as the advantages of this method. DRMs do not correspond precisely to rafts in living cells. For this reason, finding a protein enriched in DRMs does not prove that it was in rafts in the living cell. Furthermore, the fraction of a protein found in DRMs provides no quantitative information about the fraction of the protein originally in rafts. In fact, DRMs may be isolated from membranes that did not even contain rafts before detergent extraction. DRM-association is useful because it reflects a high-inherent affinity of a protein for the ordered membrane state found in rafts. Treatments that affect the DRM-association of a protein can thus be inferred to affect its raft affinity. Current models suggest that rafts may form in a regulated manner, often associated with clustering of membrane proteins or lipids, during processes such as signal transduction. DRM-association is a read-out of whether a protein is likely to associate with rafts that form under these conditions.
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References
London, E. and Brown, D. A. (2000) Insolubility of lipids in Triton X-100. Physical origin and relationship to sphingolipid/cholesterol domains (rafts). Biochim. Biophys. Acta (Biomembranes) 1508, 182–195.
Xu, X. and London, E. (2000) The effect of sterol structure on membrane lipid domains reveals how cholesterol can induce lipid domain formation. Biochemistry 39, 843–849.
Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilcheze, C., and London, E. (2001) Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide. J. Biol. Chem. 276, 33,540–33,546.
Megha and London, E. (2004) Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function. J. Biol. Chem. 279, 9997–10,004.
Brown, D. A. and 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.
Hooper, N. M. and Turner, A. J. (1988) Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor. Biochem. J. 250, 865–869.
Kenworthy, A. K., Petranova, N., and Edidin, M. (2000) High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol. Biol. Cell 11, 1645–1655.
Kenworthy, A. K., Nichols, B. J., Remmert, C. L., et al. (2004) Dynamics of putative raft-associated proteins at the cell surface. J. Cell Biol. 165, 735–746.
Glebov, O. O. and Nichols, B. J. (2004) Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat. Cell Biol. 6, 238–243.
Sharma, P., Varma, R., Sarasij, R. C., et al. (2004) Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116, 577–589.
Harder, T., Scheiffele, P., Verkade, P., and Simons, K. (1998) Lipid-domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141, 929–942.
Rothberg, K. G., Ying, Y.-S., Kamen, B. A., and Anderson, R. G. W. (1990) Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J. Cell Biol. 111, 2931–2938.
Pralle, A., Keller, P., Florin, E.-L., Simons, K., and Hörber, J. K. H. (2000) Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148, 997–1008.
Dietrich, C., Yang, B., Fujiwara, T., Kusumi, A., and Jacobson, K. (2002) Relationship of lipid rafts to transient confinement zones detected by single particle tracking. Biophys. J. 82, 274–284.
Heerklotz, H. (2002) Triton promotes domain formation in lipid raft mixtures. Biophys. J. 83, 2693–2701.
Drevot, P., Langlet, C., Guo, X.-J., et al. (2002) TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. EMBO J. 21, 1899–1908.
Vilhardt, F. and Van Deurs, B. (2004) The phagocyte NADPH oxidase depends on cholesterol-enriched membrane microdomains for assembly. EMBO J. 23, 739–748.
Magnani, F., Tate, C. G., Wynne, S., Williams, C., and Haase, J. (2004) Partitioning of the serotonin transporter into lipid microdomains modulates transport of serotonin. J. Biol. Chem. 279, 38,770–38,778.
Röper, K., Corbeil, D., and Huttner, W. B. (2000) Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582–592.
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.
Melkonian, K. A., Chu, T., Tortorella, L. B., and Brown, D. A. (1995) Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry 34, 16,161–16,170.
Mattingly, R. R. (2002) Mitogen-Activated Protein Kinase Signaling in Drug-Resistant Neuroblastoma Cells, in Cancer Cell Signaling: Methods and Protocols, (Terrian, D. M., ed.), Humana Press Inc., Totowa, NJ, pp. 71–84.
Korzeniowski, M., Kwiatkowska, K., and Sobota, A. (2003) Insights into the association of FcγRII and TCR with detergent-resistant membrane domains: Isolation of the domains in detergent-free density gradients facilitates membrane fragment reconstitution. Biochemistry 42, 5358–5367.
Sargiacomo, M., Sudol, M., Tang, Z., and Lisanti, M. P. (1993) Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol. 122, 789–807.
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Brown, D.A. (2007). Analysis of Raft Affinity of Membrane Proteins by Detergent-Insolubility. In: McIntosh, T.J. (eds) Lipid Rafts. Methods in Molecular Biology, vol 398. Humana Press. https://doi.org/10.1007/978-1-59745-513-8_2
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DOI: https://doi.org/10.1007/978-1-59745-513-8_2
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