Two‐Dimensional Gel Electrophoresis Using the ISO‐DALT System

Lonnie D. Adams1

1 The Upjohn Company, Kalamazoo, Michigan
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.3
DOI:  10.1002/0471142727.mb1003s20
Online Posting Date:  May, 2001
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Abstract

Two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) are combined in this unit to provide much greater resolution than either of the individual procedures. Solubilized proteins are first separated according to their isoelectric point by isoelectric focusing in a tube gel. This first dimension gel is then applied to the top of an SDS‐polyacrylamide slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are further separated on the basis of their molecular size. The ISO‐DALT system was specifically designed for running multiple high‐resolution two‐dimensional gels at one time.

     
 
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Table of Contents

  • Basic Protocol 1: First‐Dimension (Isoelectric Focusing) Gels
  • Basic Protocol 2: Second‐Dimension (Gradient) Gels
  • Alternate Protocol 1: Isoelectric Focusing of Basic and Very Acidic Proteins
  • Support Protocol 1: Sample Solubilization
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

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

  Materials
  • Urea
  • Ampholytes
  • recipe30% acrylamide/1.8% bisacrylamide
  • Nonidet P‐40 (NP‐40)
  • TEMED (N, N, N′, N′‐tetramethylethylenediamine)
  • recipe10% ammonium persulfate in H 2O
  • 0.85% phosphoric acid in H 2O
  • 10 M NaOH in H 2O
  • recipeBromphenol blue solution
  • 25‐ml, 10‐ml, 1‐ml, and 50‐µl syringes
  • Vacuum pump with cold trap
  • 0.2‐ or 0.45‐µm filter capsule (Acrodisk, Gelman Sciences)
  • Single‐edge razor blades
  • 22‐G hypodermic needle (2‐in. long)
  • 1‐dram vials
  • First‐dimension gel apparatus ( ISS or Hoefer Scientific)
NOTE: Distilled, deionized water should be used throughout this protocol.

Basic Protocol 2: Second‐Dimension (Gradient) Gels

  Materials
  • recipe30% acrylamide/0.8% bisacrylamide
  • recipeL10 buffer
  • recipeL20 buffer
  • 10% sodium dodecyl sulfate (SDS)
  • TEMED
  • recipe10% ammonium persulfate solution
  • recipeBlue glycerol (working solution)
  • Isobutyl alcohol, H 2O saturated
  • recipeDALT tank buffer salts
  • recipeEquilibration buffer
  • recipeAgarose solution
  • Protein molecular weight standards (Table 97.80.4711)
  • Gel plates
  • Gel identification tags
  • Gel casting box ( ISS or Hoefer Scientific)
  • Carpenter's level
  • Gradient maker ( ISS or Hoefer Scientific)
  • DALT tank ( ISS or Hoefer Scientific)
  • Loading lecturn ( ISS or Hoefer Scientific)
  • Nylon screen
The ISO‐DALT gradient maker and gel casting box are shown in Figure . NOTE: Distilled, deionized water should be used throughout this protocol.

Alternate Protocol 1: Isoelectric Focusing of Basic and Very Acidic Proteins

  Materials
  • Urea solubilization buffer (standard)
  • Urea solubilization buffer (for basic proteins)
  • SDS solubilization buffer
  • Dounce homogenizer
  • Pestles A and B
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, N.G. and Anderson, N.L. 1978a. Analytical techniques for cell fractionations. XXI. Two‐dimensional analysis of serum and tissue proteins: Multiple isoelectric focusing. Anal. Biochem. 85:332‐340.
   Anderson, N.L. and Anderson, N.G. 1978b. Analytical techniques for cell fractionations. XXII. Two‐dimensional analysis of serum and tissue proteins: Multiple gradient‐slab gel electrophoresis. Anal. Biochem. 85:341‐354.
   Celis, J.E. and Bravo, R., (eds.) 1984. Two‐Dimensional Gel Electrophoresis of Proteins. Academic Press, Orlando, Fla.
   Garrels, J.I., Farrar, J.T., and Burwell, C.B. 1984. The QUEST system for computer‐analyzed two‐dimensional electrophoresis of proteins. In Two‐Dimensional Electrophoresis of Proteins (J.E. Celis and R. Bravo, eds.) Academic Press, Orlando, Fla. pp. 37‐91.
   O'Farrell, P.H. 1975. High‐resolution two‐dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
Key Reference
   Tollaksen, S.A., Anderson, N.L., and Anderson, N.G. 1984. Operation of the ISO‐DALT system (ANL‐BIM‐84‐1). Argonne National Laboratory, Argonne, Ill.
   A detailed guide to the ISO‐DALT method for two‐dimensional gel electrophoresis.
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