SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE) of Proteins

Joanne M. Manns1

1 Temple University School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3M
DOI:  10.1002/9780471729259.mca03ms22
Online Posting Date:  August, 2011
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Abstract

Electrophoresis is a method by which a complex mixture of proteins can be separated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used to move charged molecules through a gel matrix by means of an electric current. This procedure is used to determine protein subunit composition, verify homogeneity of the protein sample, and purify proteins for use in other applications. The migration rate of the proteins during SDS-PAGE is determined by the pore size of the gel matrix and charge, size, and shape of the protein. In this unit, the protocol covers the casting of gels, preparation of the protein samples, staining and drying of the gels, and calculation of molecular mass of the proteins based on electrophoretic mobility. Curr. Protoc. Microbiol. 22:A.3M.1-A.3M.13. © 2011 by John Wiley & Sons, Inc.

Keywords: protein; electrophoresis; separation; SDS-PAGE; polyacrylamide

     
 
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Table of Contents

  • Basic Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

 Basic Protocol
 Materials
  • Laboratory detergent
  • 70% ethanol
  • 40% acrylamide/methyl bisacrylamide (ratio 37.5:1; see recipe)
  • 1.5 M Tris×Cl, pH 8.8 (appendix 2A)
  • 1.0 M Tris×Cl, pH 6.8 (appendix 2A)
  • 10% sodium dodecyl sulfate (SDS; appendix 2A)
  • 10% ammonium persulfate (APS; see recipe)
  • N,N,N¢,N¢-tetramethylethylenediamine (TEMED, electrophoresis grade; store at 4°C)
  • 0.1% SDS or water-saturated isobutanol
  • 5× Laemmli sample buffer (see recipe)
  • Protein standards: many types of protein standards/ molecular weight standards are available commercially; typically you may choose standards that are prestained so that you can monitor their migration through the gel
  • 10× Tris-glycine-SDS running buffer (see recipe)
  • Coomassie Blue staining solution (see recipe)
  • Destaining solution A (see recipe)
  • Destaining solution B (see recipe)
  • Gel drying solution (see recipe)
  • Glass gel-casting plates, spacers, and Teflon combs
  • 15-ml conical tubes
  • Wash bottle
  • 100°C water bath
  • Screw-cap or secure-lock microcentrifuge tubes
  • Gel-loading tips or Hamilton syringes
  • Vertical electrophoresis cell and power supply
  • Staining dish
  • Gel-drying frame (Diversified Biotech, cat. no. AB-100760)
  • Rustproof binder clips
  • Cellophane (Diversified Biotech, cat. no. AB-100762)
  • Molecular mass/Rf mobility acetate overlay calculator (see Fig. A.3M.2)
     FigureFigure A.3M.2 Example of an Rf calculator. This sheet is copied to transparency film using a paper copier and used as an overlay on the gel. When the transparency is placed on top of the gel, so that the top of the gel aligns with the top of the calculator and the dye front aligns with the bottom of the calculator, the Rf can be read directly off the overlay. Note that the calculator accommodates a range of gel lengths. The overlay should be copied at a 1:1 ratio so that the centimeter scale remains accurate. However, as long as the overlay can fit the top and bottom of the gel, the Rf numbers will be accurate.
  • Calculator or computer program to perform linear regression analysis
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Figures

  •  FigureFigure A.3M.1 SDS-PAGE of various protein samples with molecular weight standards in the first lane. The samples were loaded at the top of the gel and migrate down during electrophoresis.
    View Image
  •  FigureFigure A.3M.2 Example of an Rf calculator. This sheet is copied to transparency film using a paper copier and used as an overlay on the gel. When the transparency is placed on top of the gel, so that the top of the gel aligns with the top of the calculator and the dye front aligns with the bottom of the calculator, the Rf can be read directly off the overlay. Note that the calculator accommodates a range of gel lengths. The overlay should be copied at a 1:1 ratio so that the centimeter scale remains accurate. However, as long as the overlay can fit the top and bottom of the gel, the Rf numbers will be accurate.
    View Image
  •  FigureFigure A.3M.3 Standard protein molecular weight curves for (A) single concentration (5% and 12.5%) and (B) gradient (5% to 20%) gels. Protein standards are separated via SDS-PAGE, visualized by staining with Coomassie blue, and measured relative to the dye front to give the relative mobility (Rf). Note the single-concentration gel has a more limited range of linearity than the gradient gel. The standard curve permits the calculation of the molecular weight of an unknown by using the Rf of the unknown to predict the molecular weight.
    View Image

Videos

Literature Cited

Literature Cited
    Gallagher, S.R. 2006. One-dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2.1-10.2A.37.
    Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
    Maizel, J.V. 2000. SDS-polyacrylamide gel electrophoresis. Trends Biochem. Sci. 25:590-592.
    Raymond, S. and Weintraub, L. 1959. Acrylamide gel as a supporting medium for zone electrophoresis. Science 130:711.
    Shapiro, A.L., Viñuela, E., and Maizel, J.V. Jr. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820.
    Studier, F.W. 2000. Slab-gel electrophoresis. Trends Biochem Sci. 25:588-590.
    Weber, K. and Osborn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412.
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