Study of lipid rafts by gel filtration combined with preliminary staining with fluorescently labeled antibodies

  • M. P. KrutikovaEmail author
  • G. I. Krotov
  • V. G. Zgoda
  • A. V. Filatov


A novel procedure for detecting raft-associated proteins by gel filtration was developed. Cells were stained with fluorescently labeled protein-specific antibodies, lyzed in a nonionic detergent and gel-filtered on Sepharose 4B. Proteins were identified with the help of fluorescently labeled antibodies. Staining of cells with fluorescently labeled antibodies prior to cell lysis significantly simplified the identification procedure. The potentialities of the new approach are demonstrated in the example of several surface proteins associated with lipid rafts either constitutively or in the course of cell activation.


Free Volume Lipid Raft Cholera Toxin Supplement Series Brij 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



detergent-resistant membranes


fluorescein isothiocyanate


polyacrylamide gel


phosphate-buffered saline


cholera toxin B-subunit




phenylmethylsulfonyl fluoride


monoclonal antibody


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  1. 1.
    Singer, S.J. and Nicolson, G.L., The Fluid Mosaic Model of the Structure of Cell Membranes, Science, 1972, vol. 175, no. 4023, pp. 720–731.PubMedCrossRefGoogle Scholar
  2. 2.
    Schroeder, R., Ahmed, S.N., Zhu, Y., London, E., and Brown, D.A., Cholesterol and Sphingolipid Enhance the Triton X-100 Insolubility of Glycosylphosphatidylinositol-Anchored Proteins by Promoting the Formation of Detergent-Insoluble Ordered Membrane Domains, J. Biol. Chem., 1998, vol. 273, no. 2, pp. 1150–1157.PubMedCrossRefGoogle Scholar
  3. 3.
    Brown, R.E., Sphingolipid Organization in Biomembranes: What Physical Studies of Model Membranes Reveal, J. Cell Sci., 1998, vol. 111, no. 1, pp. 1–9.PubMedGoogle Scholar
  4. 4.
    Brown, D.A. and London, E., Functions of Lipid Rafts in Biological Membranes, Annu. Rev. Cell Dev. Biol., 1998, vol. 14, pp. 111–136.PubMedCrossRefGoogle Scholar
  5. 5.
    Simons, K. and Toomre, D., Lipid Rafts and Signal Transduction, Nat. Rev. Mol. Cell Biol., 2000, vol. 1, no. 1, pp. 31–39.PubMedCrossRefGoogle Scholar
  6. 6.
    Alonso, M.A. and Millán, J., The Role of Lipid Rafts in Signalling and Membrane Trafficking in T Lymphocytes, J. Cell Sci., 2001, vol. 114, no. 22, pp. 3957–3965.PubMedGoogle Scholar
  7. 7.
    Harris, T.J. and Siu, C.H., Reciprocal Raft-Receptor Interactions and the Assembly of Adhesion Complexes, Bioessays, 2002, vol. 24, no. 11, pp. 996–1003.PubMedCrossRefGoogle Scholar
  8. 8.
    Tsui-Pierchala, B.A., Encinas, M., Milbrandt, J., and Johnson, E.M., Jr., Lipid Rafts in Neuronal Signaling and Function, Trends Neurosci., 2002, vol. 25, no. 8, pp. 412–417.PubMedCrossRefGoogle Scholar
  9. 9.
    Suomalainen, M., Lipid Rafts and Assembly of Enveloped Viruses, Traffic, 2002, vol. 3, no. 10, pp. 705–709.PubMedCrossRefGoogle Scholar
  10. 10.
    Cinek, T. and Horejsi, V., The Nature of Large Noncovalent Complexes Containing Glycosyl-Phosphatidylinositol-Anchored Membrane Glycoproteins and Protein Tyrosine Kinases, J. Immunol., 1992, vol. 149, no. 7, pp. 2262–2270.PubMedGoogle Scholar
  11. 11.
    Simons, K. and van Meer, G., Lipid Sorting in Epithelial Cells, Biochemistry, 1988, vol. 27, no. 17, pp. 6197–6202.PubMedCrossRefGoogle Scholar
  12. 12.
    Radeva, G. and Sharom, F.J., Isolation and Characterization of Lipid Rafts with Different Properties from RBL-2H3 (Rat Basophilic Leukaemia) Cells, Biochem. J., 2004, vol. 380, no. 1, pp. 219–230.PubMedCrossRefGoogle Scholar
  13. 13.
    Taute, A., Kristin Watzig, K., Simons, D., Christiane Lohaus, C., Meyer, H.E., and Hasilik, A., Presence of Detergent-Resistant Microdomains in Lysosomal Membranes, Biochem. Biophys. Res. Comm., 2002, vol. 298, no. 1, pp. 5–9.PubMedCrossRefGoogle Scholar
  14. 14.
    van den Berg, C.W., Cinek, T., Hallett, M.B, Horejsi, V., and Morgan, B.P., CD59 Associates with Kinases in Membrane Clusters on U937 Cells and Becomes Ca2+-Signaling Competent, J. Cell Biol., 1995, vol. 131, no. 3, pp. 669–577.PubMedCrossRefGoogle Scholar
  15. 15.
    Claas, C., Stipp, C.S., and Hemler, M.E., Evaluation of Prototype Transmembrane 4 Superfamily Protein Complexes and Their Relation to Lipid Rafts, J. Biol. Chem., 2001, vol. 276, no. 11, pp. 7974–7984.PubMedCrossRefGoogle Scholar
  16. 16.
    Goding, J.W., Conjugation of Antibodies with Fluorochromes: Modifications to the Standard Methods, J. Immunol. Meth., 1976, vol. 13, no. 2, pp. 215–226.CrossRefGoogle Scholar
  17. 17.
    Wagener, C., Fenger, U., Clark, B.R., and Shively, J.E., Use of Biotin-Labeled Monoclonal Antibodies and Avidine-Peroxidase Conjugates for the Determination of Epitope Specificities in Solid-Phase Competitive Enzyme Immuno-Assay, J. Immunol. Meth., 1984, vol. 68, no. 1, pp. 269–274.CrossRefGoogle Scholar
  18. 18.
    Laemmli, U.K., Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685.PubMedCrossRefGoogle Scholar
  19. 19.
    Stone, K.L. and Williams, K.R., Enzymatic Digestion of Proteins in Solution in SDS Polyacrylamide Gels, The Protein Protocols Handbook, 2nd Ed., Totowa, W.J.M., Ed. (New Jersey: Humana Press, 2002), pp. 511–521.Google Scholar
  20. 20.
    Filatov, A.V., Krotov, G.I., Zgoda, V.G., and Volkov, Yu., Fluorescent Immunoprecipitation Analysis of Cell Surface Proteins: A Methodology Compatible with Mass-Spectrometry, J. Immunol. Meth., 2007, vol. 319, no. 1, pp. 21–33.CrossRefGoogle Scholar
  21. 21.
    Foster, L.J., de Hoog, C.L., and Mann, M., Unbiased Quantitative Proteomics of Lipid Rafts Reveals High Specificity for Signaling Factors, Proc. Natl. Acad. Sci. USA, 2003, vol. 100, no. 10, pp. 5813–5818.PubMedCrossRefGoogle Scholar
  22. 22.
    Filatov, A.V., Shmigol, I.B., Kuzin, I.I., Sharonov, G.V., and Feofanov, A.V., Resistance of Cellular Membrane Antigens to Solubilization with Triton X-100 As Marker of Their Association with Lipid Rafts-Analysis by Flow Cytometry, J. Immunol. Meth., 2003, vol. 278, no. 1, pp. 211–219.CrossRefGoogle Scholar
  23. 23.
    Brdickova, N., Brdicka, T., Andera, L., Spicka, J., Angelisova, P., Milgram, S.L., and Horejsi, V., Interaction between Two Adapter Proteins, PAG and EBP50: A Possible Link between Membrane Rafts and Actin Cytoskeleton, FEBS Lett., 2001, vol. 507, no. 2, pp. 133–136.PubMedCrossRefGoogle Scholar
  24. 24.
    Filatov, A.V., Shmigol, I.B., Sharonov, G.V., Feofanov, A.V., and Volkov, Y., Direct and Indirect Antibody-Induced TX-100 Resistance of Cell Suface Antigens, Immunol. Lett., 2003, vol. 85, no. 3, pp. 287–295.PubMedCrossRefGoogle Scholar
  25. 25.
    Cragg, M.S., Morgan, S.M., Chan, H.T., Morgan, B.P., Filatov, A.V., Johnson, P.W., French, R.R., and Glennie, M.J., Complement-Mediated Lysis by Anti-CD20 Mab Correlates with Segregation into Lipid Rafts, Blood, 2003, vol. 101, no. 3, pp. 1045–1052.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2007

Authors and Affiliations

  • M. P. Krutikova
    • 1
    Email author
  • G. I. Krotov
    • 1
  • V. G. Zgoda
    • 2
  • A. V. Filatov
    • 1
  1. 1.National Research Center “Institute of Immunology, Federal Medico-Biological Agency of Russia”MoscowRussia
  2. 2.Orekhovich Institute of Biomedical ChemistryRussian Academy of Medical SciencesMoscowRussia

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