Purification of Fibronectin

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 10.5
DOI:  10.1002/0471143030.cb1005s60
Online Posting Date:  September, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes the purification of the multifunctional adhesive glycoprotein fibronectin from plasma or of cell‐derived fibronectin from cell surfaces and from conditioned medium. Fibronectin can be used in cell adhesion and migration assays, and can be obtained in relatively high purity using simple affinity chromatography techniques. Curr. Protoc. Cell Biol. 60:10.5.1‐10.5.13. © 2013 by John Wiley & Sons, Inc.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Plasma Fibronectin
  • Basic Protocol 2: Purification of Fibronectin from the Surface of Cultured Cells
  • Alternate Protocol 1: Purification of Cellular Fibronectin from Conditioned Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Purification of Plasma Fibronectin

  Materials
  • Sepharose CL‐4B (GE Healthcare Life Sciences)
  • Column buffers A and B (see reciperecipes)
  • Gelatin‐Sepharose (GE Healthcare Life Sciences)
  • 6 M urea (ultrapure, Life Technologies) in TBS, pH 7.5 (see appendix 2A for TBS)
  • Surplus or outdated human plasma
  • 0.2 M EDTA, pH 7.0 ( appendix 2A)
  • 0.2 M PMSF (see recipe)
  • ϵ‐Amino‐n‐caproic acid (BioUltra grade, Sigma)
  • Citrate elution buffer (see recipe)
  • 0.5 M sodium phosphate, dibasic
  • Ammonium sulfate, molecular biology grade or similar (Sigma; optional)
  • Dialysis buffer (optional), e.g., TBS, DPBS ( appendix 2A), or CAPS/saline (see recipe)
  • Precolumn: 5‐cm diameter, ≥5 cm high, siliconized with Aquasil siliconizing fluid (Pierce Biotechnology)
  • Affinity column: 5‐cm diameter, ≥10 cm high, siliconized with Aquasil
  • High‐speed refrigerated centrifuge (e.g., Sorvall with GS‐3 and GSA rotors)
  • 250‐ or 500‐ml high‐speed centrifuge bottles
  • 50‐ml polycarbonate high‐speed centrifuge tubes (optional)
  • Spectrophotometer
  • Dialysis tubing (12,000 to 14,000 MWCO; Spectrapor or equivalent)
  • Additional reagents and equipment for dialysis ( appendix 3C), determining protein concentration by spectrophotometry ( appendix 3B), and SDS‐PAGE (unit 6.1)

Basic Protocol 2: Purification of Fibronectin from the Surface of Cultured Cells

  Materials
  • Dulbecco's modified Eagle's medium (DMEM)
  • 100 mM glutamine
  • 0.2 M PMSF (see recipe)
  • Hank's balanced salt solution (HBBS; appendix 2A)
  • Fibroblasts grown to dense confluence in ten 850‐cm2 tissue culture roller bottles using serum‐containing medium
  • Urea, ultrapure (Life Technologies)
  • Gelatin‐Sepharose (GE Healthcare Life Sciences)
  • Tris‐buffered saline (TBS; appendix 2A)
  • 8 M urea in TBS
  • Ammonium sulfate, molecular biology grade or similar (Sigma)
  • 0.2 M CAPS buffer, pH 11.0 (see recipe)
  • NaOH
  • CAPS/saline (see recipe)
  • Roller bottle apparatus
  • 50‐ml polycarbonate high‐speed centrifuge tubes
  • High‐speed refrigerated centrifuge (e.g., Sorvall with GS‐3 and GSA rotors)
  • 5‐ml siliconized glass or polypropylene column
  • Dialysis tubing (12,000 to 14,000 MWCO; Spectrapor or equivalent)
  • Spectrophotometer
  • 12 × 75–mm polypropylene tubes
  • Additional reagents and equipment for dialysis ( appendix 3C), determining protein concentration by spectrophotometry ( appendix 3B), and SDS‐PAGE (unit 6.1)

Alternate Protocol 1: Purification of Cellular Fibronectin from Conditioned Medium

  Additional Materials (also see protocol 2)
  • Serum‐free medium (e.g., supplemented 1:1 DMEM/Ham's F12)
  • Bovine serum albumin (BSA), crystalline (highest purity available; CalBiochem‐Millipore)
  • Two 2.5‐cm polypropylene or siliconized glass columns, 5 cm high (siliconized with Aquasil siliconizing fluid, Pierce)
  • 1‐liter cylindrical polypropylene bottles
  • 3.5‐ml polypropylene tubes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Akiyama, S.K., Olden, K., and Yamada, K.M. 1995. Fibronectin and integrins in invasion and metastasis. Cancer Metastasis Rev. 14:173‐189.
  Aota, S.I. and Yamada, K.M. 1995. Fibronectin and cell adhesion: Specificity of integrin‐ligand interaction. Adv. Enzymol. Rel. Areas Mol. Biol. 70:1‐21.
  Aota, S., Nomizu, M., and Yamada, K.M. 1994. The short amino acid sequence Pro‐His‐Ser‐Arg‐Asn in human fibronectin enhances cell‐adhesive function. J. Biol. Chem. 269:24756‐24761.
  Carnemolla, B., Balza, E., Siri, A., Zardi, L., Nicotra, M.R., Bigotti, A., and Natali, P.G. 1989. A tumor‐associated fibronectin isoform generated by alternative splicing of messenger RNA precursors. J. Cell Biol. 108:1139‐1148.
  Castellani, P., Viale, G., Dorcaratto, A., Nicolo, G., Kaczmarek, J., Querze, G., and Zardi, L. 1994. The fibronectin isoform containing the ED‐B oncofetal domain: A marker of angiogenesis. Int. J. Cancer 59:612‐618.
  Chen, L.B., Maitland, N., Gallimore, P.H., and McDougall, J.K. 1977. Detection of the large external transformation‐sensitive protein on some epithelial cells. Exp. Cell Res. 106:39‐46.
  Dessau, W., Sasse, J., Timpl, R., Jilek, F., and von der Mark, K. 1978a. Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix. J. Cell Biol. 79:342‐355.
  Dessau, W., Jilek, F., Adelman, B.C., and Hormann, H. 1978b. Similarity of antigelatin factor and cold insoluble globulin. Biochim. Biophys. Acta 533:227‐237.
  Engvall, E. and Ruoslahti, E. 1977. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int. J. Cancer 20:1‐5.
  Erickson, H.P. and Bourdon, M.A. 1989. Tenascin: An extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5:71‐92.
  Erickson, H.P. and Iglesias, J.L. 1984. A six‐armed oligomer isolated from cell surface fibronectin preparations. Nature 311:267‐269.
  Furcht, L.T., Mosher, D.F., and Wendelschafer‐Crabb, G. 1978. Immunocytochemical localization of fibronectin (LETS protein) on the surface of L6 myoblasts: Light and electron microscopic studies. Cell 13:263‐271.
  Hashimoto‐Uoshima, M., Yan, Y.Z., Schneider, G., and Aukhil, I. 1997. The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading. J. Cell Sci. 110:2271‐2280.
  Hayashi, M. and Yamada, K.M. 1983. Domain structure of the carboxyl‐terminal half of human plasma fibronectin. J. Biol. Chem. 258:3332‐3340.
  Humphries, M.J., Komoriya, A., Akiyama, S.K., Olden, K., and Yamada, K.M. 1987. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type‐specific adhesion. J. Biol. Chem. 262:6886‐6892.
  Hynes, R.O. 1973. Alteration of cell‐surface proteins by viral transformation and by proteolysis. Proc. Natl. Acad. Sci. U.S.A. 70:3170‐3174.
  Hynes, R.O. 1990. Fibronectin. Springer‐Verlag, New York.
  Jaffe, E.A. and Mosher, D.F. 1978. Synthesis of fibronectin by cultured human endothelial cells. J. Exp. Med. 147:1779‐1791.
  Komoriya, A., Green, L.J., Mervic, M., Yamada, S.S., Yamada, K.M., and Humphries, M.J. 1991. The minimal essential sequence for a major cell type‐specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine‐aspartic acid‐valine. J. Biol. Chem. 266:15075‐15079.
  Kornblihtt, A.R., Umezawa, K., Vibe‐Pedersen, K., and Baralle, F.E. 1985. Primary structure of human fibronectin: Differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 4:1755‐1759.
  Lightner, V.A. and Erickson, H.P. 1990. Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion. J. Cell Sci. 95:263‐277.
  Manabe, R., Ohe, N., Maeda, T., Fukuda, T., and Sekiguchi, K. 1997. Modulation of cell‐adhesive activity of fibronectin by the alternatively spliced EDA segment. J. Cell Biol. 139:295‐307.
  Miekka, S.I., Ingham, K.C., and Menache, D. 1982. Rapid methods for isolation of human plasma fibronectin. Thrombosis Res. 27:1‐14.
  Mosher, D.F. 1989. Fibronectin. Academic Press, New York.
  Pankov, R. and Yamada, K.M. 2002. Fibronectin at a glance. J. Cell Sci. 115:3861‐3863.
  Pierschbacher, M.D. and Ruoslahti, E. 1984. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309:30‐33.
  Potts, J.R. and Campbell, I.D. 1994. Fibronectin structure and assembly. Curr. Opin. Cell Biol. 6:648‐655.
  Ruoslahti, E., Vaheri, A., Kuusela, P., and Linder, E. 1973. Fibroblast surface antigen: A new serum protein. Biochim. Biophys. Acta 322:352‐358.
  Scher, B.M., Mistry, S.J., Haque, N.S., and Scher, W. 2010. Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells. Exp. Cell Res. 316:2402‐2413.
  Speziale, P., Visai, L., Rindi, S., and Poto, A.D. 2008. Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography. Nat. Protoc. 3:525‐533.
  Yamada, K.M. and Kennedy, D.W. 1984. Dualistic nature of adhesive protein function: Fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J. Cell Biol. 99:29‐36.
  Yamada, K.M. and Olden, K. 1978. Fibronectins—adhesive glycoproteins of cell surface and blood. Nature 275:179.
  Yamada, K.M. and Weston, J.A. 1974. Isolation of a major cell surface glycoprotein from fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 71:3492‐3496.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library