Electroelution of Proteins from Stained Gels

John A. Smith1

1 Merck Sharp & Dohme, Rahway, New Jersey
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.8
DOI:  10.1002/0471142735.im0808s46
Online Posting Date:  February, 2002
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Abstract

In this procedure an SDS‐polyacrylamide gel containing electrophoresed proteins is stained with Coomassie brilliant blue, the gel band of interest is excised, and the stained protein is electrophoretically eluted from the minced gel. After removal of most of the salt and sodium dodecyl sulfate (SDS) by dialysis, the protein is precipitated to remove remaining detergent and salt and then analyzed.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • SDS–polyacrylamide gel containing electrophoresed proteins (units 8.4 8.6)
  • recipeStaining solution
  • recipeDestaining solution
  • recipeSoaking buffer
  • 10% (w/v) dithiothreitol (DTT)
  • recipeElution buffer
  • recipeDialysis buffer
  • Acetonitrile or acetone, reagent‐grade
  • Electroelution apparatus (CBS Scientific)
  • recipeSpectrapor 6 dialysis membrane (Spectrum; see reagents and solutions)
  • Peristaltic pump, two‐channel
  • Pasteur pipet, bent and straight
  • 50‐µl syringe with bent, flat‐tipped needle
  • Constant voltage power supply
  • Additional reagents and equipment for preparing minigels (unit 8.4) and Coomassie blue or silver staining (unit 8.9)
NOTE: Distilled, deionized water should be used throughout this protocol.
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, C.W., Baum, P.R., and Gesteland, R.F. 1973 Processing of adenovirus 2‐induced proteins. J. Virol. 12:241‐252.
   Higgins, R.C. and Dahmus, M.E. 1979. Rapid visualization of protein bands in preparative SDS‐polyacrylamide gels. Anal. Biochem. 93:257‐260.
   Hunkapiller, M.W. and Lujan, E. 1986 Purification of microgram quantities of proteins by polyacrylamide gel electrophoresis. In Methods of Protein Microcharacterization (J. Shively, ed.) pp. 89‐101. Humana Press, Clifton, N.J.
   Hunkapiller, M.W., Lujan, E., Ostrander, F., and Hood, L.E. 1983 solation of microgram quantities of protein from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 91:227‐236.
   Wilson, K.J. and Schlabach, T.D. 1987. Reversed‐phase high‐performance liquid chromatography. 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. 10.12.1‐10.12.9, Greene Publishing and Wiley‐Interscience, New York.
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