Biotinylation and Analysis of Membrane‐Bound and Soluble Proteins

M.P. Lisanti1, M. Sargiacomo2

1 Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, 2 Instituto Superiore di Sanità, Rome, Italy
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.16
DOI:  10.1002/0471142735.im0816s36
Online Posting Date:  May, 2001
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Abstract

Coupling of a biotin moiety to proteins allows sensitive detection of those proteins with a variety of commercially available avidin or streptavidin conjugates. This unit presents protocols for biotinylation of cell‐surface proteins and proteins in solution. A major advantage of these techniques is that they do not require use of radioisotopes (although use of radioiodinated streptavidin is optional for biotin detection).

     
 
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Table of Contents

  • Basic Protocol 1: Biotinylation and Detection of Cell‐Surface Proteins
  • Basic Protocol 2: Biotinylation of Proteins in Solution
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Biotinylation and Detection of Cell‐Surface Proteins

  Materials
  • Cell culture (attached or suspension) containing ≥106 cells (107 is optimal)
  • recipePBS with Ca2+ and Mg2+ (see recipe), ice‐cold
  • recipeSulfo‐NHS‐biotin (0.5 mg/ml working solution; see recipe)
  • Serum‐free culture medium containing free amino acids (e.g., RPMI 1640 or DMEM; GIBCO/BRL), ice‐cold
  • Alkaline phosphatase (AP)– or horseradish peroxidase (HRPO)–streptavidin conjugate (e.g., Zymed)
  • Additional reagents and equipment for immunoprecipitation (optional; unit 8.3), SDS‐PAGE (unit 8.4), and protein blotting and visualization of AP and HRPO conjugates with appropriate substrates (unit 8.10)

Basic Protocol 2: Biotinylation of Proteins in Solution

  Materials
  • 1.5 mg/ml protein sample in PBS (freshly prepared or stored in 200‐µl aliquots at −20°C)
  • recipeSulfo‐NHS‐biotin (200 mg/ml stock solution; see recipe)
  • Serum‐free culture medium (e.g., RPMI 1640 or DMEM; GIBCO/BRL), ice‐cold
  • Sephadex G‐25 columns (Isolabs; bed volume 1.7 ml, void volume 0.68 ml, and maximum sample volume 0.4 ml)
  • PBS ( appendix 2A)
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Figures

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Literature Cited

Literature Cited
   Gottardi, C. and Caplan, M.J. 1993. Delivery of the Na+‐K+ ATPase in polarized cells. Science 260:552‐554.
   Graeve, L., Drikamer, K., and Rodriguez‐Boulan, E. 1989. Polarized endocytosis by MDCK cells transfected with functional chicken liver asialo‐glycoprotein receptor. J. Cell Biol. 109:2809‐2816.
   Krzeminski, K.A., Hammerton, R., Mays, R., Wollner, D., and Nelson, W.J. 1993. Delivery of the Na‐K ATPase in polarized cells. Science 260:554‐556.
   Lantz, M.L. and Holmes, L.H. 1995. Improved nonradioactive cell surface labeling technique for immunoprecipitation. Biotechniques 18:58‐60.
   Le Bivic, A., Real, F.X., and Rodriguez‐Boulan, E. 1989. Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line. Proc. Natl. Acad. Sci. U.S.A. 86:9313‐9317.
   Le Bivic, A., Quaroni, A., Nichols, B., and Rodriguez‐Boulan, E. 1990a. Biogenetic pathways of plasma membrane proteins in Caco‐2, a human intestinal epithelial cell line. J. Cell Biol. 111:1351‐1361.
   Le Bivic, A., Sambuy, Y., Mostov, K., and Rodriguez‐Boulan, E. 1990b. Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells. J. Cell Biol. 110:1533‐1539.
   Lisanti, M.P. and Rodriguez‐Boulan, E. 1991. Polarized sorting of GPI‐linked proteins in epithelia and membrane microdomains. Cell Biol. Int. Rep. 15:1023‐1049 (special issue on GPI).
   Lisanti, M.P., Sargiacomo, M., Graeve, L., Saltiel, A.R., and Rodriguez‐Boulan, E. 1988. Polarized apical distribution of glycosyl‐phosphatidylinositol anchored proteins in a renal epithelial cell line. Proc. Natl. Acad. Sci. U.S.A. 85:9557‐9561.
   Lisanti, M.P., Le Bivic, A., Sargiacomo, M., and Rodriguez‐Boulan, E. 1989. Steady‐state distribution and biogenesis of endogenous MDCK glycoproteins: Evidence for intracellular sorting and polarized cell surface delivery. J. Cell Biol. 109:2117‐2127.
   Lisanti, M.P., Caras, I.W., Gilbert, T., Hanzel, D., and Rodriguez‐Boulan, E. 1990. Vectorial apical delivery and slow endocytosis of a glycolipid‐anchored fusion protein in transfected MDCK cells. Proc. Natl. Acad. Sci. U.S.A. 87:7419‐7423.
   Sargiacomo, M., Lisanti, M.P., Graeve, L., LeBivic, A., and Rodriguez‐Boulan, E. 1989. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J. Membr. Biol. 107:277‐286.
Key References
   Lisanti et al., 1988. See above.
  Original references detailing use of sulfo‐NHS‐biotin to measure cell‐surface distribution of antigens in cultured epithelial cells. Includes critical control experiments demonstrating that biotinylation is confined to cell surface.
   Sargiacomo et al., 1989. See above.
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