Two‐Dimensional Gel Electrophoresis

Sandra Harper1, Jacek Mozdzanowski1, David Speicher1

1 The Wistar Institute, Philadelphia, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.4
DOI:  10.1002/0471140864.ps1004s11
Online Posting Date:  May, 2001
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Abstract

Two‐dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two‐dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS‐PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two‐dimensional gels, and two‐dimensional protein databases.

     
 
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Table of Contents

  • Basic Protocol 1: High‐Resolution Equilibrium Isoelectrofocusing in Tube Gels
  • Support Protocol 1: Conducting pH Profile Measurements
  • Alternate Protocol 1: Nonequilibrium Isoelectrofocusing of Very Acidic Proteins
  • Alternate Protocol 2: Nonequilibrium Isoelectrofocusing of Basic Proteins
  • Basic Protocol 2: Isoelectrofocusing Using Immobilized pH Gradient Gel Strips
  • Support Protocol 2: Electrophoresis on Immobilized pH Gradient Gels
  • Support Protocol 3: Casting an Immobiline Gel
  • Support Protocol 4: Preparing Tissue Culture Cell Extracts for Isoelectrofocusing
  • Support Protocol 5: Preparing Proteins in Tissue Samples
  • Basic Protocol 3: Second‐Dimension Electrophoresis of IEF Tube Gels
  • Basic Protocol 4: Second‐Dimension Electrophoresis of IPG Gels
  • Support Protocol 6: Preparing Molecular Weight Standards for Two‐Dimensional Gels
  • Alternate Protocol 3: Diagonal Gel Electrophoresis (Nonreducing/ Reducing Gels)
  • Support Protocol 7: Using Two‐Dimensional Protein Databases
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: High‐Resolution Equilibrium Isoelectrofocusing in Tube Gels

  Materials
  • Chromic acid, in acid‐resistant container
  • Urea (ultrapure)
  • recipe30% acrylamide/0.8% bisacrylamide (see recipe)
  • recipe20% (w/v) Triton X‐100 (see recipe)
  • Ampholytes (e.g., pH 3‐10/2D; ESA)
  • TEMED(N,N,N′,N′‐tetramethylethylenediamine)
  • recipe2.5% (w/v) ammonium persulfate (see recipe; prepare immediately before use)
  • recipe8 M urea (see recipe; prepare immediately before use)
  • recipe0.1 M orthophosphoric acid (H 3PO 4; see recipe)
  • 0.1 M NaOH ( appendix 2E; make fresh daily)
  • recipeLysis buffer (see recipe)
  • Protein samples to be analyzed
  • recipeEquilibration buffer (see recipe)
  • 2‐Mercaptoethanol
  • Isoelectrofocusing apparatus (e.g., Protean II xi 2D from Bio‐Rad or equivalent) with glass tubes, casting stand, buffer chambers, rubber grommets, and plugs
  • 37°C water bath
  • 110°C oven
  • 10‐ml syringe equipped with filter capsule (0.22 or 0.45 µm, e.g., Costar µStar LB)
  • 10‐ml syringe equipped with blunt needle [e.g., 20‐G × 6 in. (15 cm) or 18‐G × 6 in. (15 cm)]
  • Large glass cylinder sealed at bottom with Parafilm (optional, for hydrostatic pressure casting method only)
  • 2000‐V power supply
  • 60‐ml syringe
  • Metal or plastic scoop
  • Dry ice pellets

Support Protocol 1: Conducting pH Profile Measurements

  • 10% (w/v) ammonium persulfate (prepare immediately before use)
  • Concentrated sulfuric acid (used in lower chamber electrode solution)
  • Ampholytes, pH 2‐11 (used in upper chamber electrode solution)

Alternate Protocol 1: Nonequilibrium Isoelectrofocusing of Very Acidic Proteins

  Materials
  • Urea (ultrapure)
  • CHAPS or Triton X‐100
  • Pharmalyte 3‐10, 4‐6.5, and/or 8‐10.5 soluble ampholytes (see Table 10.4.1; Hoefer Pharmacia)
  • Ampholine pH 6‐8 (Hoefer Pharmacia)
  • DTT (dithiothreitol)
  • Bromphenol blue
  • Precast Immobiline DryStrips (Hoefer Pharmacia)
  • DryStrip cover fluid (Hoefer Pharmacia)
  • Immobiline DryStrip kit (Hoefer Pharmacia) including:
    •  Cathode electrode
    •  Anode electrode
    •  Sample cup bar
    •  Tray
    •  Sample cups
    •  Immobiline strip aligner
    •  IEF electrode strips
    •  Sample application pieces
    •  Instruction manual
  • Protein sample to be analyzed
  • recipeLysis buffer (see recipe)
  • Immobiline DryStrip reswelling tray (Hoefer Pharmacia)
  • Forceps
  • Filter paper
  • Glass plate
  • Flatbed electrophoresis unit (Hoefer Pharmacia Multiphor II or equivalent)
  • Recirculating cooling water bath
  • Power supply (minimum capacity of 3000 to 3500 V)
  • Petri dishes
  • Additional reagents and equipment for protein detection by staining (unit 10.5) and/or for electroblotting (unit 10.7, optional)

Alternate Protocol 2: Nonequilibrium Isoelectrofocusing of Basic Proteins

  • Precast DryPlate gel (Hoefer Pharmacia)
  • Repel‐Silane (Hoefer Pharmacia)
  • Paraffin oil
  • Protein samples to be analyzed
  • Reswelling Cassette kit (Hoefer Pharmacia) including:
    •  125 × 260 × 3–mm glass plate with 0.5‐mm U frame
    •  125 × 260 × 3–mm glass plate
    •  Silicone tubing
    •  Pinchcock
    •  Clamps
    •  20‐ml syringe
  • Roller (Hoefer Pharmacia)
  • Whatman no. 1 filter paper
  • Flatbed electrophoresis unit (Hoefer Pharmacia Multiphor II)
  • 10° or 15°C cooling water bath
  • Electrode strips
  • Sample applicator strip or sample application pieces
  • Power supply (minimum capacity 3000 to 3500 V)
  • Additional reagents and equipment for protein detection by staining (unit 10.5) and for electroblotting (unit 10.7; optional)

Basic Protocol 2: Isoelectrofocusing Using Immobilized pH Gradient Gel Strips

  • GelBond PAG film (Hoefer Pharmacia)
  • Immobiline solutions (Hoefer Pharmacia)
  • 2.5% (v/v) glycerol
  • Gradient maker
  • Orbital shaker
  • Additional materials and equipment for rehydrating immobilized pH gradient gels (see protocol 5)

Support Protocol 2: Electrophoresis on Immobilized pH Gradient Gels

  Materials
  • Cell culture flasks containing cells of interest
  • PBS with proteolysis inhibitors ( recipePBS/I buffer; see recipe)
  • Dry ice/ethanol (optional, for freezing samples)
  • recipeTris/SDS buffer (see recipe)
  • BCA protein assay kit (Pierce)
  • recipeDNase and RNase solution (see recipe)
  • 20% (w/v) SDS ( appendix 2E)
  • 2‐Mercaptoethanol
  • Urea (ultrapure)
  • recipeLysis buffer (see recipe)
  • 50‐ml centrifuge tube
  • Centrifuge with rotor (e.g., Beckman JS‐4.2), 4°C
  • 1‐ to 2‐ml cryovials
  • Microcentrifuge, 4°C
  • Sonicator with microtip
  • 0.2‐µm microcentrifuge filter units (e.g., Millipore Ultrafree‐MC filter units)

Support Protocol 3: Casting an Immobiline Gel

  Materials
  • Tissue samples
  • recipeLysis buffer (see recipe)
  • Dounce homogenizer or equivalent
  • Ultracentrifuge and rotor (e.g., Beckman Ti70), 2°C

Support Protocol 4: Preparing Tissue Culture Cell Extracts for Isoelectrofocusing

  Materials
  • recipe2% (w/v) agarose (see recipe)
  • recipeEquilibration buffer (see recipe)
  • Isoelectrofocusing gels containing protein samples to be analyzed (see protocol 1)
  • Piece of agarose containing molecular weight standards (see protocol 12)
  • Beveled glass plates
  • Boiling water bath
  • Metal or plastic scoop
  • Additional reagents and equipment for linear and gradient Laemmli gels (unit 10.1)

Support Protocol 5: Preparing Proteins in Tissue Samples

  • recipeDryStrip equilibration solutions 1 and 2 (see recipes; prepare fresh in step )
  • Immobiline IPG DryStrip with focused protein (see protocol 5)
  • Platform shaker

Basic Protocol 3: Second‐Dimension Electrophoresis of IEF Tube Gels

  Materials
  • Molecular weight standards (Table 97.80.4711)
  • 1× SDS sample buffer (unit 10.1)
  • recipe2% (w/v) agarose (see recipe)
  • Boiling water bath
  • Glass tubes (3‐mm inner diameter)
  • Plastic or metal tray

Basic Protocol 4: Second‐Dimension Electrophoresis of IPG Gels

  • Separating and stacking gel solutions (see 97.80.4711)
  • 1× SDS sample buffer without reducing agents (unit 10.1)
  • recipeReducing buffer (see recipe)
  • recipe1.5% (w/v) agarose in reducing buffer (see recipe; optional, for securing first‐dimension gel on second‐dimension gel)
  • Two‐dimensional comb (optional)
  • Additional reagents and equipment for casting tube gels (see protocol 1), SDS‐PAGE (unit 10.1), and protein staining (unit 10.5)
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Figures

  •   FigureFigure 10.4.1 Marking orientation of a precast IPG gel when only a portion of the gel is used.
  •   FigureFigure 10.4.2 Casting the second‐dimension gel and loading the IEF gel. (A) The stacking gel solution should reach to the upper edge of the beveled plate, and then the gel solution has to be overlaid with a minimum volume of water. The water will stay on the surface because of surface tension. (B) After polymerization, the gel is mounted on the central cooling core of the electrophoresis unit, and the equilibrated IEF gel is placed on top of the polymerized stacking gel. Excess buffer is removed, and the IEF gel is overlaid with hot agarose/equilibration buffer mixture. After the agarose solidifies, the upper electrophoresis chamber is filled with buffer.

Videos

Literature Cited

Literature Cited
   Anderson, N.G. and Anderson, N.L. 1978a. Two‐dimensional analysis of serum and tissue proteins: Multiple isoelectrofocusing. Anal. Biochem. 85:331‐340.
   Anderson, N.L. and Anderson, N.G. 1978b. Two‐dimensional analysis of serum and tissue proteins: Multiple gradient‐slab gel electrophoresis. Anal. Biochem. 85:341‐354.
   Celis, J.E., Rasmussen, H.H., Leffers, H., Madsen, P., Honore, B., Gesser, B., Dejgaard, K., and Vandekerckhove, J. 1991. Human cellular protein patterns and their link to genome DNA sequence data: Usefulness of two‐dimensional gel electrophoresis and microsequencing. FASEB J. 5:2200‐2208.
   Dunbar, B. 1987. Troubleshooting and artifacts in two‐dimensional polyacrylamide gel electrophoresis. In Two‐Dimensional Electrophoresis and Immunological Techniques (B.S. Dunbar, ed.) pp. 173‐195. Plenum, New York.
   Garrels, J.I. 1979. Two‐dimensional gel electrophoresis and computer analysis of proteins synthesized by cloned cell lines. J. Biol. Chem. 54:7961‐7977.
   Garrels, J.I. 1989. The QUEST system for quantitative analysis of two‐dimensional gels. J. Biol. Chem. 264:5269‐5282.
   Garrels, J.I. and Franza, B.R., Jr. 1989. The REF52 protein database: Methods of database construction and analysis using the QUEST system and characterizations of protein patterns from proliferating and quiescent REF52 cells. J. Biol. Chem. 264:5283‐5298.
   Goverman, J. and Lewis, K. 1991. Separation of disulfide‐bonded polypeptides using two‐imensional diagonal gel electrophoresis. Methods 3:125‐127.
   Hochstrasser, D.F., Harrington, M.C., Hochstrasser, A.C., Miller, M.J., and Merril, C.R. 1988. Methods for increasing the resolution of two‐dimensional protein electrophoresis. Anal. Biochem. 173:424‐435.
   O'Farrell, P.H. 1975. High resolution two‐dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
   Strahler, J.R. and Hanash, S.M. 1991. Immobilized pH gradients: Analytical and preparative use. Methods 3:109‐114.
   Young, D.A., Voris, B.P., Maytin, E.V., and Colbert, R.A. 1983. Very high resolution two‐dimensional electrophoretic separation of proteins on giant gels. Methods Enzymol. 91:190‐214.
Key Reference
   Hochstrasser et al., 1988. See above.
  Discusses methods for improving and troubleshooting two‐dimensional separation.
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