Purification of Vitronectin

Steven K. Akiyama1

1 National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.6
DOI:  10.1002/0471143030.cb1006s60
Online Posting Date:  September, 2013
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This unit describes the purification of extracellular vitronectin from plasma or serum by using heparin‐affinity chromatography. First, the plasma is depleted of fibronectin plus other heparin‐ and Sepharose‐binding proteins and treated with urea to activate the heparin‐binding activity of vitronectin, which is subsequently bound to a heparin affinity column and eluted. The resulting vitronectin should be ∼98% pure. Curr. Protoc. Cell Biol. 60:10.6.1‐10.6.7. © 2013 by John Wiley & Sons, Inc.

Keywords: extracellular matrix; cell biology; affinity purification; adhesion

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1:

  • Sepharose CL‐4B (GE Healthcare Life Sciences)
  • Phosphate/saline/EDTA (see recipe)
  • Heparin‐Sepharose (GE Healthcare Life Sciences)
  • 2 M NaCl in phosphate/saline/EDTA (see recipe)
  • ∼400 ml (1 to 2 units) citrate‐anticoagulated human plasma; alternatively serum can be used
  • 0.2 M EDTA, pH 7.0 ( appendix 2A)
  • 0.2 M phenylmethylesulfonyl fluoride (PMSF; unit 10.5)
  • 95% ethanol, ice cold
  • Ultrapure urea
  • 8 M urea in phosphate/saline/EDTA (see recipe)
  • 8 M urea/2 M NaCl in phosphate/saline/EDTA (see recipe)
  • 2‐mercaptoethanol
  • 8 M urea/0.3 M NaCl in phosphate/saline/EDTA (see recipe)
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A)
  • 20‐ and 50‐ml (minimum) 2.5‐cm‐diameter polypropylene or siliconized glass columns
  • 600‐ and 1000‐ml polypropylene beakers
  • Refrigerated centrifuge
  • Polypropylene centrifuge tubes
  • Whatman no. 2 filter paper
  • 10‐ml polypropylene tubes
  • Quartz spectrophotometer cuvettes siliconized with Aquasil (Pierce Biotechnology)
  • UV‐Vis spectrophotometer
  • Dialysis tubing (12,000 to 14,000 MWCO)
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Literature Cited

Literature Cited
  Barnes, D.W. and Silnutzer, J. 1983. Isolation of human serum spreading factor. J. Biol. Chem. 258:12548‐12552.
  Dahlback, B. and Podack, E.R. 1985. Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group. Biochemistry 24:2368‐2374.
  Hayashi, M., Akama, T., Kono, I., and Kashiwagi, H. 1985. Activation of vitronectin (serum spreading factor) binding of heparin by denaturing agents. J. Biochem. 98:1135‐1138.
  Hayman, E.G., Pierschbacher, M.D., Ohgren, Y., and Ruoslahti, E. 1983. Serum spreading factor (vitronectin) is present at the cell surface and in tissues. Proc. Natl. Acad. Sci. U.S.A. 80:4003‐4007.
  Kitagaki‐Ogawa, H., Yatohgo, T., Izumi, M., Hayashi, M., Kashiwagi, H., Matsumoto, I., and Seno, N. 1990. Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains. Biochim. Biophys. Acta 1033:49‐56.
  Miyazaki, K., Hamano, T., and Hayashi, M. 1992. Heat and autoclave resistance of cell‐spreading activity of vitronectin. Biochim. Biophys. Acta 1159:215‐222.
  Nagano, Y., Hamano, T., Nakashima, N., Ishikawa, M., Miyazaki, K., and Hayashi, M. 1992. Yolk vitronectin. Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding, and bound carbohydrate. J. Biol. Chem. 267:24863‐24870.
  Preissner, K.T. 1991. Structure and biological role of vitronectin. Annu. Rev. Cell Biol. 7:275‐310.
  Skurzynski, S. 2010. One‐step purification of vitronectin from human plasma by affinity chromatography on phage‐displayed peptides. Acta Biochim. Pol. 57:89‐93.
  Tomasini, B.R. and Mosher, D.F. 1991. Vitronectin. Prog. Hemost. Thromb. 10:269‐305.
  Yatohgo, T., Izumi, M., Kushiwagi, H., and Hayashi, M. 1988. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct. Funct. 13:281‐292.
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