Two‐Dimensional Gel Electrophoresis

Lonnie D. Adams1, Sean R. Gallagher2

1 The Upjohn Company, Kalamazoo, Michigan, 2 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.5
DOI:  10.1002/0471142735.im0805s68
Online Posting Date:  November, 2005
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Abstract

Two‐dimensional gel electrophoresis is the combination of two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first‐dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS‐slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two‐dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the support protocol.

     
 
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Table of Contents

  • Basic Protocol 1: First‐Dimension (Isoelectric‐Focusing) Gels
  • Support Protocol 1: Solubilization and Preparation of Proteins in Tissue Samples
  • Basic Protocol 2: Second‐Dimension Gels
  • Alternate Protocol 1: Isoelectric Focusing of Very Basic Proteins Using NEPHGE
  • Alternate Protocol 2: Isoelectric Focusing of Very Acidic Proteins Using NEPHGE
  • Alternate Protocol 3: Two‐Dimensional Minigels
  • Alternate Protocol 4: Two‐Dimensional (2‐D) Electrophoresis with Immobilized pH Gradients
  • Support Protocol 2: Rehydration of IPG Strips Using the Immobiline Drystrip Reswelling Tray
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: First‐Dimension (Isoelectric‐Focusing) Gels

  Materials
  • Urea (ultrapure)
  • 30% acrylamide/1.8% bisacrylamide (see recipe)
  • Ampholytes, pH 4 to 8 (Bio‐Rad, Serva, Invitrogen, Sigma‐Aldrich)
  • Nonidet P‐40 (NP‐40)
  • TEMED (N,N,N′,N′ tetramethylethylenediamine)
  • 10% ammonium persulfate (see recipe)
  • 0.085% phosphoric acid (see recipe)
  • 0.02 M NaOH (see recipe)
  • Protein samples (see protocol 2)
  • Concentrated bromphenol blue (see recipe)
  • Chromic acid cleaning solution (see recipe)
  • 1.0‐ to 3.0‐mm‐inner‐diameter glass gel tubes (∼1.5 in. longer than the width of the second‐dimension gel; 4‐ to 6‐mm outer diameter)
  • 2.5‐ to 3.0‐cm‐inner‐diameter gel‐casting glass tube, ∼2 cm shorter than gel tubes
  • Small vacuum flask
  • 50‐µl, 1‐ml, and 20‐ml syringes
  • 0.2‐ or 0.45‐µm filter capsule (Acrodisk; Gelman)
  • Single‐edge razor blade
  • Rubber grommets
  • Tube cell (Bio‐Rad, Topac, CBS Scientific, Scie‐Plas)
  • 22‐G hypodermic needle (2‐in. long)
  • 200‐µl pipettor tip
  • 1‐dram gel vials
NOTE: Distilled, deionized water should be used throughout this protocol.

Support Protocol 1: Solubilization and Preparation of Proteins in Tissue Samples

  Materials
  • Tissue samples
  • SDS or urea solubilization buffer (see reciperecipes)
  • Dounce homogenizer with pestles A and B
  • 200‐µl centrifuge tubes
  • Beckman 42.2‐Ti rotor (or equivalent)

Basic Protocol 2: Second‐Dimension Gels

  Materials
  • 30% acrylamide/0.8% bisacrylamide (see recipe for acrylamide/bisacrylamide solutions)
  • Gel buffer (see recipe)
  • 10% (w/v) SDS
  • TEMED
  • 10% ammonium persulfate (see recipe)
  • Isobutyl alcohol, H 2O‐saturated
  • Stacking gel buffer (optional; see recipe)
  • First‐dimension gel (see protocol 1)
  • Equilibration buffer (see recipe)
  • Hot 0.5% and 1% agarose (see recipe; keep in boiling water bath)
  • Protein molecular weight standards (Table 97.80.4711; available as kits Bio‐Rad or Amersham Biosciences)
  • SDS solubilization buffer (see recipe)
  • Reservoir buffer (see recipe), prechilled to 10° to 20°C
  • Coolant (from running tap water or circulating refrigerated water bath)
  • Gel plates, one long and one short
  • 1.5‐mm spacers (∼14 cm × 14 cm × 0.75 mm)
  • Casting stand
  • Gel identification tag (e.g., typed consecutive numbers on filter paper)
  • Nylon screen
  • 5 × 15–cm glass plate
  • PROTEAN II electrophoresis cell (Bio‐Rad)

Alternate Protocol 1: Isoelectric Focusing of Very Basic Proteins Using NEPHGE

  • Ampholytes, pH 2 to 11 (Serva)
  • 0.01 M phosphoric acid, deaerated
  • 4 M urea, deaerated
To analyze very basic proteins, the procedure is the same as described in protocol 1 for first‐dimension gels with the following exceptions in the indicated steps:

Alternate Protocol 2: Isoelectric Focusing of Very Acidic Proteins Using NEPHGE

  • Ampholytes, pH 2.5 to 4 (Pharmacia LKB)
  • Concentrated sulfuric acid
  • Water, deaerated

Alternate Protocol 3: Two‐Dimensional Minigels

  Materials
  • Protein sample for analysis
  • Sample solution: default sample solution, hydrophobic sample solution, or tissue sample solution (see recipe)
  • Tissue homogenization solution (see recipe)
  • Rehydration stock solution (see recipe)
  • IPG buffer or Pharmalyte (same range as the IPG strip; Amersham Biosciences)
  • Cleaning solution (e.g., Ettan IPGphor Strip Holder Cleaning Solution; Amersham Biosciences)
  • IPG Dry Strip Cover Fluid (Amersham Biosciences)
  • Vertical gel for SDS‐PAGE (Basic Protocol in unit 8.4)
  • SDS equilibration buffer (see recipe)
  • SDS electrophoresis buffer (unit 8.4)
  • Molecular weight markers (unit 8.4)
  • Agarose sealing solution (see recipe)
  • Isoelectric focusing system (Ettan IPGphor, Amersham Biosciences; PROTEAN IEF System, Bio‐Rad)
  • IPG strips (Amersham Biosciences)
  • Toothbrush
  • Platform rocker
  • 100°C heating block
  • Thin plastic ruler
  • Additional reagents and equipment for second‐dimension gel electrophoresis (see protocol 3)

Alternate Protocol 4: Two‐Dimensional (2‐D) Electrophoresis with Immobilized pH Gradients

  • Immobiline DryStrip Reswelling Tray (Amersham Biosciences)
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Figures

  •   FigureFigure 8.5.1 Assembly of gel tubes in casting tube.
  •   FigureFigure 8.5.2 Assembly of second‐dimension gel.
  •   FigureFigure 8.5.3 Coomassie‐stained proteins separated by two‐dimensional gel electrophoresis. In this gel, a broad range pH gradient (pH 3.5 to 9) was used for isoelectric focusing in the first dimension, and a 10 to 20% gradient SDS/polyacrylamide gel was used in the second dimension.

Videos

Literature Cited

   Anderson, N.L. 1988. Two‐Dimensional Electrophoresis Operation of the ISO‐DALT (R) System. Large Scale Biology Press, Washington, D.C.
   Berkelman, T. and Stenstedt, T. 1998. 2‐D Electrophoresis. Principles and Methods. Amersham Biosciences, San Francisco.
   Bjellqvist, B., Ek, K., Righetti, P.G., Gianazza, E., Görg, A., Westermeier, R., and Postel, W. 1982. Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications. J. Biochem. Biophys. Methods 6:317‐339.
   Dunbar, B.S. 1987. Two‐Dimensional Electrophoresis and Immunological Techniques. Plenum, New York.
   Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., and Weiss, W. 2000. The current state of two‐dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037‐1053.
   Hirano, H. 1989. Microsequence analysis of winged bean seed proteins electroblotted from two‐dimensional gel. J. Prot. Chem. 8:115‐130.
   Lilley, K.S. 2002. Protein profiling using two‐dimensional difference gel electrophoresis (2‐D DIGE). In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 22.2‐22.14. John Wiley & Sons, Hoboken, N.J.
   Ochs, D.C., McConkey, E.H., and Sammons, D.W. 1981. Silver stains for proteins in polyacrylamide gels: A comparison of six methods. Electrophoresis. 2:304‐307.
   O'Farrell, P.H. 1975. High‐resolution two‐dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
   Patterson, S.D. 1998. Protein identification and characterization by mass spectrometry. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.22.1‐10.22.24. John Wiley & Sons, Hoboken, N.J.
   Westermeier, R., Postel, W., Weser, J., and Görg, A. 1983. High‐resolution two‐dimensional electrophoresis with isoelectric focusing in immobilized pH gradients. J. Biochem. Biophys. Methods 8:321‐330.
   Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K., Golaz, O., Sanchez, J.C., Yan, J.X., Gooley, A.A., Hughes, G., Humphery‐Smith, I., Williams, K.L., Hochstrasser, D.F. 1996. From proteins to proteomes: Large scale protein identification by two‐dimensional electrophoresis and amino acid analysis. Biotechnology (N.Y.) 14:61‐65.
Key References
   Berkelman and Stenstedt, 1998. See above.
  Manual for the GE/Amersham family of 2‐D electrophoresis equipment; details the background and protocols for use of immobilized pH gradient (IPG) strips in high‐resolution analysis.
   Celis, J.E. and Bravo, R. (eds.) 1984. Two‐Dimensional Gel Electrophoresis of Proteins. Academic Press, San Diego, Calif.
  Provides detailed information on two‐dimensional gels and their applications.
   Dunbar, 1987. See above.
  A comprehensive guide to two‐dimensional electrophoresis. It covers basic principles of electrophoresis, gives instructions for performing two‐ dimensional electrophoresis and associated procedures such as protein detection, photography, and preparation of antibodies from proteins excised from two‐dimensional gels.
   Garfin, D. and Heerdt, L. (eds.) 2000. 2D Electrophoresis for Proteomics. A Methods and Product Manual. Bio‐Rad Laboratories, Inc., Hercules, Calif.
  Manual for the Bio‐Rad family of 2‐D electrophoresis equipment; details the background and protocols for use of immobilized pH gradient (IPG) strips in high‐resolution analysis.
   Görg et al., 2000. See above.
  An important update on the latest developments in 2‐D electrophoresis.
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