One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels

Hidde L. Ploegh1

1 Massachusetts Institute of Technology, Cambridge
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.2
DOI:  10.1002/0471140864.ps1002s00
Online Posting Date:  May, 2001
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Abstract

This unit describes isoelectric focusing (IEF) of polypeptides in urea‐containing polyacrylamide slab gels as a result of a pH gradient created by ampholytes (small charged organic molecules) in the gel in response to an electric field. Separation on the basis of isoelectric point produces bands of polypeptides that can be electrotransferred to suitable membranes for further analysis, for example, by staining or immunodetection. If samples have been radiolabeled, they can be visualized by fluorography. This unit contains support protocols detailing special conditions for application of denaturing IEF slab gels for electrotransfer and fluorography in which a scintillator is incorporated into the gel after completion of the run to facilitate detection of radioactively labeled polypeptides.

     
 
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Table of Contents

  • Basic Protocol 1: Isoelectric Focusing in Slab Gels Under Denaturing Conditions
  • Support Protocol 1: Electroblotting from Denaturing Isoelectric‐ Focusing Slab Gels
  • Support Protocol 2: Fluorography of Denaturing Isoelectric‐Focusing Slab Gels
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Isoelectric Focusing in Slab Gels Under Denaturing Conditions

  Materials
  • Urea (any high‐grade urea will usually prove satisfactory)
  • 10% (v/v) Triton X‐100
  • recipeAcrylamide stock solution (see recipe)
  • Ampholytes pH 5‐7
  • Ampholytes pH 3.5‐10
  • 10% (w/v) ammonium persulfate (store 500‐µl aliquots of freshly prepared stock at −20°C)
  • TEMED (N,N,N′‐N′‐tetramethylenediamine)
  • 20 mM H 3PO 4
  • Protein samples to be analyzed
  • recipeIEF solubilization buffer (see recipe)
  • Overlay solution: recipeIEF solubilization buffer diluted 1:3 (v/v) with H 2O
  • 20 mM NaOH (freshly made, degassed immediately prior to use)
  • Electrophoresis apparatus (e.g., Studier type or equivalent) with glass plates, spacers, clamps, casting stand, and buffer chambers
  • Side‐arm flask
  • Comb
  • Power supply (capable of delivering at least 1000 V and 10 W total power)
  • Hamilton syringe or mechanical pipet
CAUTION: Acrylamide is a neurotoxin; wear gloves when handling solutions.

Support Protocol 1: Electroblotting from Denaturing Isoelectric‐ Focusing Slab Gels

  Materials
  • DMSO
  • recipeDMSO/PPO solution (see recipe)
  • Polyacrylamide gel containing polypeptides of interest
  • Film cassette
  • Film
  • Intensifying screen
CAUTION: Solutions of PPO in DMSO readily permeate the skin,causing the PPO to precipitate subcutaneously. It is therefore imperative that gloves be worn at all times while handling DMSO/PPO solutions.
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Figures

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Literature Cited

Literature Cited
   Benaroch, P., Yilla, M., Raposo, G., Ito, K., Miwa, K., Geuze, H.J., and Ploegh, H.L. 1995. How MHC Class II molecules reach the endocytic pathway. EMBO J. 14:37‐49.
   Eichholtz, T.E., Vossebeld, P., Van Overveld, M., and Ploegh, H.L. 1992. Activation of protein kinase C accelerates internalization of transferrin receptor but not major histocompatibility complex class I molecules, independent of their phosphorylation status. J. Biol. Chem. 267:22490‐22495.
   Garrels, J.I. 1989. The QUEST system for quantitative analysis of two‐dimensional gels. J. Biol. Chem. 264:5269‐5282.
   Heemels, M.T., Schumacher, T.N.M., Wonigeit, K., and Ploegh, H.L. 1993. Different peptides are translocated by allelic variants of the transporter associated with antigen presentation (TAP). Science 262:2059‐2063.
   O'Farrell, P.H. 1975. High resolution two‐dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007‐4021.
Key References
   Benaroch, et al., 1995. See above.
  These contain representative examples of good quality separations of membrane proteins attained by the IEF methods described here.
   Eichholtz et al., 1992. See above.
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