SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE)

Sean R. Gallagher1

1 UVP, LLC, Upland, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 7.3
DOI:  10.1002/9780470089941.et0703s06
Online Posting Date:  September, 2012
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Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), investigate subunit compositions, verify homogeneity of protein samples, and purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). Support protocols cover the casting of gels, calculation of molecular mass using the electrophoretic mobility of a protein, and purification of SDS by recrystallization. Curr. Protoc. Essential Lab. Tech. 6:7.3.1‐7.3.28. © 2012 by John Wiley & Sons, Inc.

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

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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method
  • Support Protocol 1: Casting a Gel for Use in Denaturing Discontinuous Electrophoresis
  • Support Protocol 2: Calculating Molecular Mass
  • Support Protocol 3: Recrystallizing SDS
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: The Laemmli Gel Method

  • Protein sample to be analyzed: 1 to 50 µg in <20 µl (depending on sample complexity) when staining with Coomassie blue; 10‐ to 100‐fold less protein when silver staining (0.01 to 5 µg in <20 µl)
  • 2× or 6× SDS sample buffer (see reciperecipes)
  • 1× SDS sample buffer: dilute from 2× or 6× stock
  • Protein molecular‐weight‐standards mixture (Table 7.3.1 and Fig. )
  • Polyacrylamide gel, purchased precast (e.g., Table 7.3.2) or self‐cast ( protocol 2)
  • 1× SDS electrophoresis buffer (see reciperecipes)
  • 100°C and 56°C water bath
  • Screw‐cap microcentrifuge tube
  • 100°C heating block
  • Electrophoresis apparatus (see Table 7.3.2), small format with 100‐mA capability constant‐current power supply (allowing running of two gels simultaneously), including clamps, glass plates, casting stand, and buffer chambers (e.g., XCell SureLock Mini‐Cell, Life Technologies/Invitrogen; Mini‐Protean, Bio‐Rad; or SE 250 10‐cm unit, Hoefer)
  • Absorbent paper
CAUTION: The voltages and currents used during electrophoresis are dangerous and potentially lethal. It is extremely important to read the section entitled Safety Considerations before performing any electrophoresis.

Support Protocol 1: Casting a Gel for Use in Denaturing Discontinuous Electrophoresis

  • Detergent, laboratory quality (e.g., Alconox or RBS‐35; Pierce)
  • 30% acrylamide/0.8% bisacrylamide solution (see recipe), room temperature
  • 1× and 4× Tris·Cl (pH 8.8)/SDS (see recipe), room temperature
  • 10% ammonium persulfate (APS), prepare fresh
  • N, N, N′, N′‐tetramethylethylenediamine (TEMED)
  • Water‐saturated isobutyl alcohol (see recipe)
  • Glass plates (part of electrophoresis apparatus; see ( protocol 1):
  • Laboratory marker (e.g., Sharpie)
  • 0.75‐, 1.0‐, or 1.5‐mm Teflon spacers
  • Casting stand
  • 25‐ml Erlenmeyer side‐arm flask with solid rubber stopper
  • Vacuum pump with cold trap
  • Teflon comb (same thickness as spacers) with 1, 3, 5, 10, 15, or 20 teeth

Support Protocol 2: Calculating Molecular Mass

  • Gel containing separated proteins ( protocol 1)
  • Molecular mass/R f acetate overlay calculator (Fig. )
  • Calculator or analysis program for performing linear regression

Support Protocol 3: Recrystallizing SDS

  • SDS
  • 100% ethanol, room temperature
  • Water, 55°C
  • Activated charcoal, Norit 1 (Sigma)
  • 100% reagent‐grade ethanol, –20°C
  • 55°C water bath
  • Buchner funnel and Whatman no. 5 paper
  • Coarse‐frit (porosity A) sintered‐glass funnel
  • Vacuum source
  • Desiccator containing charged phosphorous pentoxide (P 2O 5)
  • Dark bottle
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Literature Cited

   Adams, L.D. and Gallagher, S. 2004. Two‐dimensional gel electrophoresis. Curr. Protoc. Mol. Biol. 67:10.4.1‐10.4.23.
   Dhugga, K.S., Waines, J.G., and Leonard, R.T. 1988. Correlated induction of nitrate uptake and membrane polypeptides in corn roots. Plant Physiol. 87:120‐125.
   Gallagher, S.R. 1999. One‐dimensional electrophoresis using nondenaturing conditions. Curr. Protoc. Mol. Biol. 47:10.2B.1‐10.2B.11.
   Gallagher, S.R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallagher, S.R. and Leonard, R.T. 1987. Electrophoretic characterization of a detergent‐treated plasma membrane fraction from corn roots. Plant Physiol. 83:265‐271.
   Hunkapiller, M.W., Lujan, E., Ostrander, F., and Hood, L.E. 1983. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 91:227‐236.
   Jovin, T.M. 1973. I. Steady‐state moving‐boundary systems formed by different electrolyte combinations. Biochemistry 12:871‐879.
   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   Matsudaira, P.T. and Burgess, D.R. 1978. SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal. Biochem. 87:386‐396.
   Okajima, T., Tanabe, T., and Yasuda, T. 1993. Nonurea sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with high‐molarity buffers for the separation of proteins and peptides. Anal. Biochem. 211:293‐300.
   Ornstein, L. 1964. Disc electrophoresis. I. Background and theory. Ann. N.Y. Acad. Sci. 121:321‐349.
   Ploegh, H.L. 1995. One‐dimensional isoelectric focusing of proteins in slab gels. Curr. Protoc. Protein Sci. 0:10.2.1‐10.2.8.
   Schagger, H. and von Jagow, G. 1987. Tricine‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:368‐379.
   Scopes, R.K. 1995. Overview of protein purification and characterization. Curr. Protoc. Protein Sci. 0:1.1.1‐1.1.6.
   Takano, E., Maki, M., Mori, H., Hatanaka, N., Marti, T., Titani, K., Kannagi, R., Ooi, T., and Murachi, T. 1988. Pig heart calpastatin: Identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. Biochemistry 27:1964‐1972.
   Weber, K., Pringle, J.R., and Osborn, M. 1972. Measurement of molecular weights by electrophoresis on SDS‐acrylamide gel. Methods Enzymol. 26:3‐27.
Key Reference
   Hames, B.D. and Rickwood, D. (eds.) 1990. Gel Electrophoresis of Proteins: A Practical Approach, 2nd Ed. Oxford University Press, New York.
  An excellent book describing gel electrophoresis of proteins.
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