Biotin Labeling and Quantitation of Cell‐Surface Proteins

Diane N. Turvy1, Janice S. Blum1

1 Indiana University School of Medicine, Indianapolis, Indiana
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.7
DOI:  10.1002/0471142735.im1807s36
Online Posting Date:  May, 2001
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Abstract

Cell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Endocytosis in Nonadherent Cell Lines
  • Alternate Protocol 1: Detection of Endocytosis in Adherent Cell Lines
  • Support Protocol 1: ELISA for Quantitating Biotin‐Labeled Proteins
  • Support Protocol 2: Immunoblotting for Detection of Biotinylated Proteins
  • Support Protocol 3: Quantitating Surface Half‐Life of Proteins Using Biotin Labeling
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Detection of Endocytosis in Nonadherent Cell Lines

  Materials
  • Sulfosuccinimidyl‐2‐(biotinamido) ethyl‐1,3‐dithiopropionate (sulfo‐NHS‐S‐S‐biotin; Pierce)
  • HBSS ( appendix 2A)
  • Nonadherent cells
  • HBSS with 5 mM Tris, pH 7.4
  • 0.45 M sucrose in HBSS (optional)
  • 0.3 mM primaquine solution in complete RPMI‐1 ( appendix 2A) with 5 mM HEPES
  • recipeGlutathione stripping solution (see recipe)
  • recipeLysis buffer (see recipe)
  • 0.5 M Tris⋅Cl, pH 7.4 ( appendix 2A)
  • Additional reagents and equipment for quantitation of biotin‐labeled protein (see protocol 3Support Protocols 1 to protocol 53)

Alternate Protocol 1: Detection of Endocytosis in Adherent Cell Lines

  • 150 × 25–mm tissue culture dishes

Support Protocol 1: ELISA for Quantitating Biotin‐Labeled Proteins

  Materials
  • Purified monoclonal antibodies (mAbs) specific for the receptors or antigens of interest
  • Anti‐transferrin receptor mAb (Pharmingen; Boehringer Mannheim)
  • PBS ( appendix 2A2)
  • 5% (v/v) FBS in PBS
  • Irrelevant isotype‐matched antibody (optional)
  • PBS‐T: PBS containing 0.05% Tween 20
  • Biotin‐labeled cell lysate (see protocol 1 or protocol 2)
  • 2.5 µg/ml HRP‐avidin solution: horseradish peroxidase–conjugated avidin in PBS containing 10% (v/v) heat‐inactivated FBS
  • 2,2′‐Azino‐di(3‐ethylbenzthiazoline sulfonate) (ABTS; BioFX Laboratories)
  • 96‐well EIA/RIA high protein–binding plates
  • Microtiter plate reader with 405‐nm filter

Support Protocol 2: Immunoblotting for Detection of Biotinylated Proteins

  Materials
  • Biotin‐labeled cell lysate (see protocol 1 or protocol 2)
  • Mouse antibodies specific for cell‐surface receptors or antigens of interest
  • Protein A–Sepharose or protein G–Sepharose beads precoated with rabbit anti–mouse IgG (unit 8.3)
  • recipeTBS (see recipe)
  • recipe2× reducing SDS‐PAGE sample buffer (see recipe)
  • 1% (w/v) BSA/0.05% (v/v) Tween 20/PBS (see appendix 2A for PBS)
  • 0.5 µg/ml horseradish peroxidase (HRP)–streptavidin in PBS
  • Enhanced chemiluminescence reagents (ECL; Amersham Pharmacia Biotech)
  • Electroblotting apparatus
  • Additional reagents and equipment for immunoprecipitation (unit 8.3), SDS PAGE (unit 8.4), and electroblotting (unit 8.10)

Support Protocol 3: Quantitating Surface Half‐Life of Proteins Using Biotin Labeling

  • Sulfo‐N‐hydroxysuccinimidobiotin (Sulfo‐NHS‐biotin; Pierce)
  • 0.45‐µm filter unit
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Figures

Videos

Literature Cited

Literature Cited
   Ajioka, S. and Kaplan, J. 1986. Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors. Proc. Natl. Acad. Sci. U.S.A 83:6445.
   Amigorena, S., Webster, P., Drake, J., Newcomb, J., Cresswell, P., and Mellman, I. 1995. Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles. J. Exp. Med 181:1729.
   Anderson, G.W., Zimmerman, J., and Callahan, F. 1964. The uses of esters of N‐hydroxysuccinimide in peptide synthesis. J. Am. Chem. Soc. 86:1839.
   Bos, C.R., Shank, S.L., and Snider, M.D. 1995. Role of clathrin‐coated vesicles in glycoprotein transport from the cell surface to the golgi complex. J. Biol. Chem. 270:665.
   Brachet, V., Raposo, G., Amigorena, S., and Mellman, I. 1997. Ii controls the transport of major histocompatibility complex class II to and from lysosomes. J. Cell Biol. 137:51.
   Bretscher, M.S. and Lutter, R. 1988. A new method for detecting endocytosed proteins. EMBO J. 7:4087.
   Daukas, G. and Zigmond, S.H. 1985. Inhibition of receptor‐mediated but not fluid‐phase endocytosis in polymorphonuclear leukocytes. J.Cell Biol. 101:1673.
   Geuze, H.J., Slot, J.W., and Schwartz, A.L. 1987. Membranes of sorting organelles display lateral heterogeneity in receptor distribution. J. Cell Biol. 104:1715.
   Gruenberg, J. and Howell, K.E. 1989. Membrane traffic in endocytosis: Insights from cell‐free assays. Annu. Rev. Cell Biol. 5:453.
   Heuser, J.E. and Anderson, R.G.W. 1989. Hypertonic media inhibit receptor‐mediated endocytosis by blocking clathrin‐coated pit formation. J. Cell Biol. 108:389.
   Larkin, J.M., Brown, M.S., Goldstein, J.L., and Anderson, R.G.W. 1983. Depletion of intracellular potassium arrests coated pit formation and receptor‐mediated endocytosis in fibroblasts. Cell 33:273.
   Luna, E.J. 2000. Biotinylation of proteins in solution and on cell surfaces In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds.) pp. 3.6.1‐3.6.15. John Wiley & Sons, New York
   Marmorstein, A.D., Boniha, V.L., Chiflet, S., Neill, J.M., and Rodriguez‐Boulan, E. 1996. The polarity of the plasma membrane protein RET‐PE2 in retinal pigment epithelium is developmentally regulated. J. Cell Sci. 109:3025.
   Milliken, G.A. and Johnson, D.E. 1984. Analysis of Messy Data, Vol. 1, pp. 36‐40. Van Nostrand–Reinhold New York.
   Pearse, B.M. and Robinson, M.S. 1990. Clathrin, adaptors and sorting. Annu. Rev. Cell Biol. 6:151.
   Pinet, V., Vergelli, M., Martin, R., Bakke, O., and Long, E.O. 1995. Antigen presentation mediated by recycling of surface HLA‐DR molecules. Nature 375:603.
   Reid, P.A. and Watts, C. 1990. Cycling of cell surface MHC glycoproteins through primaquine‐sensitive intracellular compartments. Nature 346:655.
   Ryder, E.F. and Robakiewicz, P. 2000. Statistics for the molecular biologist: Group comparisons. In Current Protocols in Molecular Biology (R. Brent, R.E. Kington, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3I.1‐A.3I.22. John Wiley & Sons, New York
   Schwartz, A.L., Bolognesi, A., and Fridovich, S.E. 1984. Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells. J. Cell Biol. 98:732.
   Stoorvogel, W., Oorschot, V., and Geuze, J. 1996. A novel class of clathrin‐coated vesicles budding from endosomes. J. Cell Biol. 132:21.
   Tse, D.B. and Pernis, B. 1984. Spontaneous internalization of class I major histocompatibility complex molecules in T lymphoid cells. J. Exp. Med. 159:193
   Turvy, D. and Blum, J. 1998. Detection of biotinylated cell surface receptors and MHC molecules in a capture ELISA: A rapid assay to measure endocytosis. J. Immunol. Methods 212:9.
   Volz, B., Orberger, G., Porwoll, S., Hauri, H.P., and Tauber, R. 1995. Selective reentry of recycling cell surface glycoproteins to the biosynthetic pathway in human hepatocarcinoma HepG2 cells. J. Cell Biol. 130:537.
Key References
   Pernis, B. 1985. Internalization of lymphocyte membrane components. Immunol. Today 6:45.
  Overview of membrane protein recycling in lymphocytes, including the first description of recycling MHC molecules in lymphocytes.
   Reid and Watts, 1990. See above.
  First description of MHC class II endocytosis and recycling.
   Turvy and Blum, 1998. See above.
  First publication of this methodology, describing the use of biotin labeling to quantitate cell‐surface protein endocytosis.
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