Agarose Gel Electrophoresis of Proteins

Dennis M. Krizek1, Margaret E. Rick1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.7
DOI:  10.1002/0471143030.cb0607s15
Online Posting Date:  August, 2002
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Abstract

This unit describes electrophoretic separation and identification of large proteins from a complex protein mixture. Agarose gel is utilized for the electrophoretic matrix, and detection of proteins is accomplished by transfer of the proteins to a membrane that is probed with specific antibodies and chemiluminescence reagents. Alternatively the protein can be detected in the gel using radiolabeled antibodies and autoradiography.

     
 
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Table of Contents

  • Basic Protocol 1: Agarose Gel Electrophoresis and Blotting with Immunodetection
  • Alternate Protocol 1: Agaraose Gel Electrophoresis with In‐Gel Antibody Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Agarose Gel Electrophoresis and Blotting with Immunodetection

  Materials
  • SeaKem HGT(P) agarose (Bio Whittaker or equivalent)
  • 1× electrophoresis buffer, 4°C (see recipe)
  • Protein samples
  • 2× sample buffer (see recipe)
  • 0.25× transfer buffer without methanol (see recipe)
  • recipeBlocking buffer (unit 6.2) containing 5% (w/v) nonfat dry milk, fresh
  • Antibodies:
  •  Primary: rabbit anti‐vWF (Dako)
  •  Secondary: donkey horseradish peroxidase–linked anti‐rabbit Ig (Amersham Pharmacia)
  • ECL Western Blotting Analysis system (Amersham Pharmacia)
  • Aluminum foil
  • Boiling waterbath (optional)
  • Horizon 20‐25 horizontal electrophoresis apparatus (Life Technologies) or equivalent
  • Teflon comb (e.g., 1 × 9–mm, 20‐well)
  • Pipet with fine tip or equivalent
  • 0.45‐µm Immobilon‐P polyvinylidene fluoride (PVDF) membrane (Millipore)
  • Additional reagents and equipment for protein transfer to membranes and immunoblotting (unit 6.2)

Alternate Protocol 1: Agaraose Gel Electrophoresis with In‐Gel Antibody Analysis

  Materials
  • In‐gel sample buffer, fresh (see recipe)
  • Borate saline buffer (BSB; see recipe)
  • Sample
  • 0.5% (w/v) bromphenol blue in H 2O
  • Isopropanol
  • Agarose gel buffer (see recipe)
  • SeaKem HGT(P) agarose (FMC/BioWhittaker Molecular)
  • Agarose running buffer (see recipe)
  • Fixing buffer (see recipe)
  • Blocking buffer, in‐gel (see recipe)
  • 125I‐labeled rabbit anti‐human vWF polyclonal antibody (Dako #A0082): radiolabel using protocol of choice and immunopurify (Hoyer and Shainoff, ; also see unit 7.10)
  • 2% (w/v) human IgG (see recipe)
  • High‐salt wash buffer (see recipe)
  • 12 × 75–mm polypropylene tube
  • 12.5 × 26.0 × 0.3–cm glass plate (Amersham Pharamacia Biotech)
  • 12.5 × 24.0–cm spacer plate with adherent 0.5‐mm spacers (Amersham Pharmacia Biotech)
  • 12.4 × 25.8–cm GelBond film (Amersham Pharmacia Biotech)
  • Flexiclamps (Amersham Pharmacia Biotech)
  • 20‐ml syringe
  • 60°C oven
  • Aluminum foil
  • Gelman Delux electrophoresis chamber (Gelman Sciences) or equivalent
  • 104 × 253–mm paper electrophoresis electrode wicks (Amersham Pharmacia Biotech)
  • Flattened no. 2 cork borer
  • Forceps, fine
  • Horizontal rotary mixer
  • Forced hot‐air dryer (optional)
  • Kodak X‐Omatic film cassette with Lanex screens and film
CAUTION: When working with radioactivity, take apptopriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by your local radiations safety officer (also see appendix 1D).
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Figures

Videos

Literature Cited

Literature Cited
   Aronson, D.L., Krizek, D.M., and Rick, M.E. 2001. A rapid assay for the vWF protease. Thrombosis and Hemostasis 85:184‐185.
   Hoyer, L.W. and Shainoff, J.R. 1980. Factor VIII‐related protein circulates in normal human plasma as high molecular weight multimers. Blood 55:1056‐1059.
   Krizek, D.M. and Rick, M.E. 2000. A rapid method to visualize von Willebrand factor multimers using agarose gel electrophoresis, immunolocalization, and luminographic detection. Thrombosis Research 97:457‐62
   Krizek, D.M. and Rick, M.E. 2001. Clinical application of a rapid method using agarose gel electrophoresis and western blotting to evaluate von Willebrand factor protease activity. Electrophoresis 22:946‐949.
   Peacock, A.C. and Dingman, W.C. 1968. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose‐acrylamide composite gels. Biochemistry 7:668‐674.
   Rick, M.R. 2002. Hemophilia and von Willebrand disease. In UpToDate Clinical Reference Library, Release 9.1 (B.D. Rose, ed.) UpToDate, Wellesley, MA.
   Shainoff, J.R. 1993. Electrophoresis and direct immunoprobing on glyoxal agarose. In Advances in Electrophoresis, Vol. 6 (A. Chrambach, M.J. Dunn, and B.J. Radola, eds.) pp. 65‐176. VCH Publishers, New York.
   Shainoff, J.R., Urbanic, D.A., and DiBello, P.M. 1991. Immunoelectrophoretic characterizations of the cross‐linking of fibrinogen and fibrin by factor XIIIa and tissue transglutaminase. Identification of a rapid mode of hybrid alpha‐/gamma‐chain cross‐linking that is promoted by the gamma‐chain cross‐linking. J. Biol. Chem. 266:6429‐6437.
Key References
   Krizek and Rick, 2000. See above.
  This original paper of the procedure and use of immunoblotting and chemiluminescence for ararose gel electrophoresis provides the background and reasons for the development of this assay in the clinical laboratory setting.
   Hoyer and Shainoff, 1980. See above.
  This paper outlines the original “in‐gel” procedure for the electrophoresis of very high molecular weight proteins and provides examples of its usefulness in understanding the structure of von Willebrand factor.
   Shainoff, 1993. See above
  This reference provides general background and the rationale for the use of modified ararose for the separation and identification of (large) proteins by electrophoresis.
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