One‐Dimensional SDS Gel Electrophoresis of Proteins

Sean R. Gallagher1

1 UVP, LLC, Upland, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.1
DOI:  10.1002/0471140864.ps1001s68
Online Posting Date:  April, 2012
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Abstract

One‐dimensional gel electrophoresis of proteins provides information about the molecular size, amount, and purity of a protein sample. Separated proteins can be recovered from polyacrylamide gels for subsequent characterization by a variety of secondary techniques, such as mass spectrometry to determine post‐translational modifications and the amino acid sequence. In addition, one‐dimensional electrophoresis is the standard first step in immunoblotting and immunodetection. Protein separations in vertical slab gels are performed in a variety of formats. Most recently, small format minigels are typical due to their ease of use, low relative cost, and ready commercial availability. Larger gels provide more separation area and thus better resolution for complex samples and continue to be in use for specialized analysis. Curr. Protoc. Protein Sci. 68:10.1.1‐10.1.44. © 2012 by John Wiley & Sons, Inc.

Keywords: electrophoresis; SDS‐PAGE; polyacrylamide; PAGE; minigel; protein; molecular weight; precast gels

     
 
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Table of Contents

  • Introduction
  • Electricity and Electrophoresis
  • Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method
  • Alternate Protocol 1: Electrophoresis in Tris‐Tricine Buffer Systems
  • Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers
  • Alternate Protocol 3: Continuous SDS‐PAGE
  • Alternate Protocol 4: Casting and Running Ultrathin Gels
  • Support Protocol 1: Casting Multiple Single‐Concentration Gels
  • Alternate Protocol 5: Separation of Proteins on Gradient Gels
  • Support Protocol 2: Casting Multiple Gradient Gels
  • Basic Protocol 2: Electrophoresis in Single‐Concentration Minigels
  • Support Protocol 3: Preparing Multiple Gradient Minigels
  • Support Protocol 4: Calculating Molecular Mass
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method

  Materials
  • Separating and stacking gel solutions (Table 10.1.1)
  • H 2O‐saturated isobutyl alcohol
  • 1× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.1.1)
  • Protein sample, on ice
  • 2× and 1× SDS sample buffer (see recipe)
  • Protein molecular weight standards (Tables 10.1.2 and 10.1.3)
  • 6× SDS sample buffer (see recipe; optional)
  • 1× SDS electrophoresis buffer (see recipe)
  • Electrophoresis apparatus: e.g., Protean II 16‐cm cell (Bio‐Rad) or SE 600/400 16‐cm unit (Hoefer) with clamps, glass plates, casting stand, and buffer chambers
  • 0.75‐mm spacers
  • 0.45‐µm filters (used in stock solution preparation)
  • 25‐ml Erlenmeyer side‐arm flasks
  • Vacuum pump with cold trap
  • 0.75‐mm Teflon comb with 1, 3, 5, 10, 15, or 20 teeth
  • Screw‐top microcentrifuge tubes (recommended)
  • 25‐ or 100‐µl syringe with flat‐tipped needle
  • Constant‐current power supply (see Electricity and Electrophoresis above)

Alternate Protocol 1: Electrophoresis in Tris‐Tricine Buffer Systems

  • Separating and stacking gel solutions (Table 10.1.5)
  • 2× tricine sample buffer (see recipe)
  • Peptide molecular weight standards (Table 10.1.6)
  • Cathode buffer (see recipe)
  • Anode buffer (see recipe)
  • Coomassie blue G‐250 staining solution (see recipe)
  • 10% (v/v) acetic acid
  • 50‐ml Erlenmeyer side‐arm flasks

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

  • Separating and stacking gel solutions (Table 10.1.7)
  • 2× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.1.1)
  • 2× SDS electrophoresis buffer (see recipe)

Alternate Protocol 3: Continuous SDS‐PAGE

  • Separating gel solution (Table 10.1.8)
  • 2× and 1× phosphate/SDS sample buffer (see recipe)
  • 1× phosphate/SDS electrophoresis buffer (see recipe)

Alternate Protocol 4: Casting and Running Ultrathin Gels

  • 95% (v/v) ethanol
  • Gel Bond (FMC BioProducts) cut to a size slightly smaller than the gel plate dimensions
  • Glue stick
  • Ink roller (available from art supply stores)
  • Combs and spacers (0.19 to 0.5 mm; sequencing gel spacers and combs can be cut to fit)

Support Protocol 1: Casting Multiple Single‐Concentration Gels

  • Separating and stacking gels for single‐concentration gels (Table 10.1.9)
  • Multiple gel caster (Bio‐Rad, Hoefer)
  • 100‐ml disposable syringe and flat‐tipped needle
  • Extra plates and spacers
  • 14 × 14–cm acrylic blocks or polycarbonate sheets
  • 250‐ and 500‐ml side‐arm flasks (used in gel preparation)
  • Long razor blade or plastic wedge (Wonder Wedge, Hoefer)
  • Resealable plastic bags

Alternate Protocol 5: Separation of Proteins on Gradient Gels

  • Light and heavy acrylamide gel solutions (Table 10.1.10)
  • Bromphenol blue (optional; for checking practice gradient)
  • 10% ammonium persulfate (prepare fresh)
  • TEMED
  • Gradient maker (30 to 50 ml, Hoefer SG30 or SG50; or 30 to 100 ml, Bio‐Rad 385)
  • Tygon tubing with micropipet tip
  • Peristaltic pump (optional; e.g., Markson A‐13002, A‐34040, or A‐34105 minipump)
  • Whatman 3MM filter paper

Support Protocol 2: Casting Multiple Gradient Gels

  • Plug solution (see recipe)
  • Light and heavy acrylamide gel solutions for multiple gradient gels (Table 10.1.11)
  • TEMED
  • H 2O‐saturated isobutyl alcohol
  • Multiple gel caster (Bio‐Rad, Hoefer)
  • Peristaltic pump (25 ml/min)
  • 500‐ or 1000‐ml gradient maker (Bio‐Rad, Hoefer)
  • Tygon tubing

Basic Protocol 2: Electrophoresis in Single‐Concentration Minigels

  Materials
  • Minigel vertical gel unit (Hoefer Mighty Small SE 250/280 or Bio‐Rad Mini‐Protean II) with glass plates, clamps, and buffer chambers
  • 0.75‐mm spacers
  • Multiple gel caster (Hoefer SE‐275/295 or Bio‐Rad Mini‐Protean II multicasting chamber)
  • Acrylic plate (Hoefer SE‐217 or Bio‐Rad 165‐1957) or polycarbonate separation sheet (Hoefer SE‐213 or Bio‐Rad 165‐1958)
  • 10‐ and 50‐ml syringes
  • Combs (Teflon, Hoefer SE‐211A series or Bio‐Rad Mini‐Protean II)
  • Long razor blade
  • Micropipet
  • Additional reagents and equipment for standard denaturing SDS‐PAGE (see protocol 1)

Support Protocol 3: Preparing Multiple Gradient Minigels

  • Plug solution (see recipe)
  • Additional reagents and equipment for preparing gradient gels (see protocol 7)
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Figures

Literature Cited

   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. 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.
   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.
   Ploegh, H.L. 1995. One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, and P.T. Wingfield, eds.) pp. 10.2.1‐10.2.8. John Wiley & Sons, Hoboken, N.J.
   Schagger, H. and vonJagow, 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.
   See, Y.P., Olley, P.M., and Jackowski, G. 1985. The effects of high salt concentrations in the samples on molecular weight determination in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresis 8:382‐387.
   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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