Non‐Radioactive Quantification of Fibronectin Matrix Assembly

Roumen Pankov1, Kenneth M. Yamada1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.13
DOI:  10.1002/0471143030.cb1013s25
Online Posting Date:  December, 2004
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This unit provides a basic protocol for non‐radioactive determination of the rate of incorporation of biotinylated fibronectin into the insoluble matrix organized by cultured cells. The protocol provides quantification of changes in matrix assembly due to different experimental conditions with concomitant determinations of the activation state of various signaling molecules that may be involved in the process of matrix assembly. This unit also provides a support protocol for biotinylation of purified fibronectin.

Keywords: fibronectin; biotinylation; deoxycholate; solubility; phosphospecific antibodies

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Table of Contents

  • Basic Protocol 1: Quantification of Matrix Assembly Using Biotinylated Fibronectin
  • Support Protocol 1: Biotinylation of Plasma Fibronectin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Quantification of Matrix Assembly Using Biotinylated Fibronectin

  • Fibroblasts (or any cell line of interest)
  • Dulbecco's modified Eagle medium supplemented with 10% (v/v) fetal bovine serum (DMEM/10% FBS; appendix 2A)
  • Biotinylated fibronectin (see protocol 2)
  • PBS ( appendix 2A), ice cold
  • DOC extraction buffer (see recipe)
  • 2× SDS sample buffer ( appendix 2A)
  • 1 M NaF
  • 0.1 M sodium orthovanadate solution ( appendix 1B)
  • 10 mM leupeptin ( appendix 1B)
  • 25 mM pepstatin A ( appendix 1B)
  • 0.2 M phenylmethanesulfonyl fluoride (PMSF; appendix 1B)
  • 2× SDS sample buffer ( appendix 2A)
  • 8% (w/v) polyacrylamide separating gels with 4% (w/v) stacking gels (unit 6.1) or commercially available pre‐cast 4% to 12% gradient gels (e.g., Novex) for SDS gel electrophoresis
  • Prestained protein molecular size standards (e.g., Novex)
  • Transfer buffer (unit 6.2)
  • Ponceau S solution (unit 6.2)
  • Tris‐buffered saline with 0.1% (v/v) Tween 20 (TTBS; appendix 2A)
  • Blocking solution: TTBS containing 5% (w/v) dry nonfat milk (TTBS/milk)
  • Streptavidin, horseradish peroxidase (HRP)‐conjugated (e.g., Jackson ImmunoResearch)
  • Enhanced chemiluminescence (ECL) detection reagent (unit 14.2)
  • Primary antibody: monoclonal anti‐vimentin (e.g., Sigma)
  • Secondary antibody: horseradish peroxidase (HRP)‐conjugated anti‐rabbit or anti‐mouse antibodies (e.g., Amersham Bioscience)
  • 35‐mm tissue culture dishes
  • Plastic cell scraper (rubber policeman)
  • 23‐G needle and 1‐ml syringe
  • 1.5‐ml microcentrifuge tubes
  • Micropipettors
  • Porous electrotransfer pads
  • Whatman 3MM filter papers cut to gel size
  • Two nitrocellulose membranes cut to gel size
  • SDS‐PAGE/transfer apparatus (e.g., Bio‐Rad, Novex)
  • Constant‐voltage/current power supply (e.g., Bio‐Rad)
  • Flat containers
  • Rocking shaker
  • Heat‐sealable plastic bags and sealer
  • Sonicator/ultrasonic processor
  • Plastic wrap
  • X‐ray film (e.g., Hyperfilm; Amersham Bioscience)
  • Tube heater (e.g., Thermomixer; Eppendorf) or boiling water bath
  • Film cassette for X‐ray film
  • X‐ray film developer
  • Additional reagents and equipment for dialysis ( appendix 3C), tissue culture (unit 1.1), SDS‐PAGE (unit 6.1), and immunoblotting (unit 6.2)
NOTE: All reagents and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a 37°C, 10% CO 2 humidified incubator. Use pre‐warmed cell culture medium for all treatments.

Support Protocol 1: Biotinylation of Plasma Fibronectin

  • Fibronectin (e.g., Sigma or purified as described in unit 10.5)
  • Bicarbonate buffer (see recipe)
  • Sulfo‐NHS‐biotin (e.g., Pierce)
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Literature Cited

Literature Cited
   Coulombe, P.A., Ma, L., Yamada, S., and Wawersik, M. 2001. Intermediate filaments at a glance. J. Cell Sci. 114:4345‐4347.
   Geiger, B., Bershadsky, A., Pankov, R., and Yamada, K.M. 2001. Transmembrane extracellular matrix–cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2:793‐805.
   McKeown‐Longo, P.J. and Mosher, D.F. 1983. Binding of plasma fibronectin to cell layers of human skin fibroblasts. J. Cell Biol. 97:466‐472.
   Wierzbicka‐Patynowski, I. and Schwarzbauer, J.E. 2002. Regulatory role for SRC and phosphatidylinositol 3‐kinase in initiation of fibronectin matrix assembly. J. Biol. Chem. 277:19703‐19708.
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