Two‐Dimensional Gel Electrophoresis

Sandra Harper1, Jacek Mozdzanowski1, David Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.4
DOI:  10.1002/0471143030.cb0604s04
Online Posting Date:  May, 2001
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Abstract

While one‐dimensional SDS‐PAGE separates proteins on the basis of size, two‐dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two‐dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two‐dimensional protein databases.

     
 
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Table of Contents

  • Basic Protocol 1: High‐Resolution Equilibrium Isoelectric Focusing in Tube Gels
  • Support Protocol 1: Conducting pH Profile Measurements
  • Alternate Protocol 1: Nonequilibrium Isoeletric Focusing of very Acidic Proteins
  • Alternate Protocol 2: Nonequilibrium Isoelectric Focusing of Basic Proteins
  • Basic Protocol 2: Isoelectric Focusing 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 Isoelectric Focusing
  • 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: Digonal 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 Isoelectric Focusing in Tube Gels

  Materials
  • Chromic acid, in acid‐resistant container
  • Urea (ultrapure)
  • 30% acrylamide/0.8% bisacrylamide (see recipe)
  • 20% (w/v) Triton X‐100 (see recipe)
  • Ampholytes (e.g., pH 3‐10/2D; ESA)
  • TEMED (N,N,N′,N′‐tetramethylethylenediamine)
  • 2.5% (w/v) ammonium persulfate (see recipe; prepare immediately before use)
  • 8 M urea (see recipe; prepare immediately before use)
  • 0.1 M orthophosphoric acid (H 3PO 4; see recipe)
  • 0.1 M NaOH (make fresh daily)
  • Lysis buffer (see recipe)
  • Protein samples to be analyzed
  • Equilibration buffer (see recipe)
  • 2‐Mercaptoethanol
  • Isoelectric focusing 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)
To analyze very acidic proteins, follow protocol 1 with these exceptions in the indicated steps:

Alternate Protocol 1: Nonequilibrium Isoeletric Focusing 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 6.4.1; Amersham Pharmacia Biotech)
  • Ampholine pH 6‐8 (Amersham Pharmacia Biotech)
  • DTT recipe(dithiothreitol)
  • Bromphenol blue
  • Precast Immobiline DryStrips (Amersham Pharmacia Biotech)
  • DryStrip cover fluid (Amersham Pharmacia Biotech)
  • Immobiline DryStrip kit (Amersham Pharmacia Biotech) 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
  • Lysis buffer (see recipe)
  • Immobiline DryStrip reswelling tray (Amersham Pharmacia Biotech)
  • Forceps
  • Filter paper
  • Glass plate
  • Flatbed electrophoresis unit (Amersham Pharmacia Biotech 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 ( appendix 3A) and/or for electroblotting (unit 6.2, optional)

Alternate Protocol 2: Nonequilibrium Isoelectric Focusing of Basic Proteins

  • Precast DryPlate gel (Amersham Pharmacia Biotech)
  • Repel‐Silane (Amersham Pharmacia Biotech)
  • Paraffin oil
  • Protein samples to be analyzed
  • Reswelling Cassette kit (Amersham Pharmacia Biotech) 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 (Amersham Pharmacia Biotech)
  • Whatman no. 1 filter paper
  • Flatbed electrophoresis unit (Amersham Pharmacia Biotech 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 ( appendix 3A) and for electroblotting (unit 6.2; optional)

Basic Protocol 2: Isoelectric Focusing Using Immobilized pH Gradient Gel Strips

  • GelBond PAG film (Amersham Pharmacia Biotech)
  • Immobiline solutions (Amersham Pharmacia Biotech)
  • 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 (PBS/I buffer; see recipe)
  • Dry ice/ethanol (optional, for freezing samples)
  • Tris/SDS buffer (see recipe)
  • BCA protein assay kit (Pierce)
  • DNase and RNase solution (see recipe)
  • recipe20% (w/v) SDS( appendix 2E)
  • 2‐Mercaptoethanol
  • Urea (ultrapure)
  • Lysis 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
  • Lysis 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 Isoelectric Focusing

  Materials
  • 2% (w/v) agarose (see recipe)
  • Equilibration buffer (see recipe)
  • Isoelectric focusing 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 6.1)

Support Protocol 5: Preparing Proteins in Tissue Samples

  • DryStrip equilibration solutions 1 and 2 (see recipe; 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 6.4.2)
    Table 6.4.2   MaterialsSize Options for Two‐Dimensional Gel Electrophoresis

    Gel type First‐dimension gel Second‐dimension gel e Purpose Comments f
    Diameter D (mm) Length L (cm) Thickness T (mm) Height H (cm)
    Microgels/minigels g <1.5 <10 <D <10 Analytical 1‐4
    <1.5 <10 >D <10 Analytical 3‐6
    >1.5 <10 <D h <10 Analytical/preparative 1,2,7,8
    Full‐size gels j <1.5 12‐18 <D 12‐18 Analytical 1‐4
    <1.5 12‐18 >D 12‐18 Analytical 3‐6
    >1.5 12‐18 <D h 12‐18 Analytical/preparative 1,2,7,8
    Giant gels k <1.5 >20 >D >20 Analytical 4‐6,9
    >1.5 >20 <D h >20 Analytical/preparative 1‐3,7

     eThe second‐dimension gel width has to be at least equal to the IEF tube gel height.
     fKey to comments: (1) tube gel cannot be placed directly on top of second‐dimension gel, and use of agarose is recommended; (2) use of stacking gel is recommended; (3) extrusion and handling are relatively difficult; (4) total protein load is limited to usually ≤50 µg for whole‐cell or tissue extracts; (5) tube gel can be placed directly on top of second‐dimension gel, and use of agarose is not necessary; (6) use of a stacking gel is not necessary; (7) total protein load capacity is relatively large; (8) extrusion and handling are relatively simple; (9) extrusion and handling are very difficult.
     gMinigel systems provide rapid separations with moderate resolution. Microgels (Phastgels) are precast gels that are slightly smaller than most minigels.
     hUse of second‐dimension gels thicker than 1.5 mm is generally not recommended owing to difficulty with either efficient staining or efficient electroblotting.
     JFull‐size gels provide resolution satisfactory for most applications.
     kGiant gels provide very good resolution. Specialized equipment is required, such as Investigator 2D (ESA), Iso‐Dalt (Hoeffer Pharmacia), or homemade giant‐size gel systems.
  • recipeSDS sample buffer (unit 6.1)
  • 2% (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 Table 97.80.4711)
  • recipeSDS sample bufferwithout reducing agents (unit 6.1)
  • Reducing buffer (see recipe)
  • 1.5% (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 6.1), and protein staining ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, N.G. and Anderson, N.L. 1978a. Two‐dimensional analysis of serum and tissue proteins: Multiple isoelectric focusing. 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, Jr., B.R. 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‐dimensional 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., See
  Discusses methods for improving and troubleshooting two‐dimensional separation.
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