One‐Dimensional SDS Gel Electrophoresis of Proteins

Sean R. Gallagher1

1 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Appendix 3F
DOI:  10.1002/0471140856.txa03fs32
Online Posting Date:  May, 2007
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Abstract

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to 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, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris‐tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.

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

     
 
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Table of Contents

  • 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 3.0.1)
  • H 2O‐saturated isobutyl alcohol
  • 1× Tris⋅Cl/SDS, pH 8.8 (dilute 4× Tris⋅Cl/SDS, pH 8.8; Table 3.0.1)
  • Protein sample, on ice
  • 2× and 1× SDS sample buffer (see recipe)
  • Protein molecular weight standards (Tables 3.0.2 and 3.0.3)
  • 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 above)

Alternate Protocol 1: Electrophoresis in Tris‐Tricine Buffer Systems

  • Separating and stacking gel solutions (Table 3.0.5)
  • 2× tricine sample buffer (see recipe)
  • Peptide molecular weight standards (Table 3.0.6)
  • 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
    Table 0.f.5   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Tricine Peptide Separating and Stacking Gels f   Recipes for Tricine Peptide Separating and Stacking Gels   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis j   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis

    SEPARATING AND STACKING GELS
    Stock solution g Separating gel Stacking gel
    30% acrylamide/0.8% bisacrylamide 9.80 ml 1.62 ml
    Tris⋅Cl/SDS, pH 8.45 10.00 ml 3.10 ml
    H 2O 7.03 ml 7.78 ml
    Glycerol 4.00 g (3.17 ml)
    10% (w/v) ammonium persulfate h 50 µl 60 µl
    TEMED 10 µl 10 µl
    Prepare separating and stacking gel solutions separately.
    • In a 50‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (Table 3.0.1), Tris⋅Cl/SDS, pH 8.45 (see reagents, below), and H 2O. Add glycerol to separating gel only. Degas under vacuum 10 to 15 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix; use immediately.
    ADDITIONAL REAGENTS USED IN GELS
    Tris⋅Cl/SDS, pH 8.45 (3.0 M Tris⋅Cl containing 0.3% SDS)
    • Dissolve 182 g Tris base in 300 ml H 2O. Adjust to pH 8.45 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter, add 1.5 g SDS, and store at 4°C up to 1 month.
    Peptide Molecular weight (Da)
    Myoglobin (polypeptide backbone) 16,950
    Myoglobin 1‐131 14,440
    Myoglobin 56‐153 10,600
    Myoglobin 56‐131 8,160
    Myoglobin 1‐55 6,210
    Glucagon 3,480
    Myoglobin 132‐153 2,510

     fThe recipes produce 30 ml of separating gel and 12.5 ml of stacking gel, which are adequate for two gels of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the Tris‐tricine buffer system of Schagger and von Jagow ( ).
     gAll reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     hBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.
    Table 0.f.6   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Tricine Peptide Separating and Stacking Gels f   Recipes for Tricine Peptide Separating and Stacking Gels   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis j   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis

    SEPARATING AND STACKING GELS
    Stock solution g Separating gel Stacking gel
    30% acrylamide/0.8% bisacrylamide 9.80 ml 1.62 ml
    Tris⋅Cl/SDS, pH 8.45 10.00 ml 3.10 ml
    H 2O 7.03 ml 7.78 ml
    Glycerol 4.00 g (3.17 ml)
    10% (w/v) ammonium persulfate h 50 µl 60 µl
    TEMED 10 µl 10 µl
    Prepare separating and stacking gel solutions separately.
    • In a 50‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (Table 3.0.1), Tris⋅Cl/SDS, pH 8.45 (see reagents, below), and H 2O. Add glycerol to separating gel only. Degas under vacuum 10 to 15 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix; use immediately.
    ADDITIONAL REAGENTS USED IN GELS
    Tris⋅Cl/SDS, pH 8.45 (3.0 M Tris⋅Cl containing 0.3% SDS)
    • Dissolve 182 g Tris base in 300 ml H 2O. Adjust to pH 8.45 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter, add 1.5 g SDS, and store at 4°C up to 1 month.
    Peptide Molecular weight (Da)
    Myoglobin (polypeptide backbone) 16,950
    Myoglobin 1‐131 14,440
    Myoglobin 56‐153 10,600
    Myoglobin 56‐131 8,160
    Myoglobin 1‐55 6,210
    Glucagon 3,480
    Myoglobin 132‐153 2,510

     JPeptide standards are commercially available from Sigma‐Aldrich. See Sigma‐Aldrich Technical Bulletin MWSDS70‐L for molecular weight markers for proteins.

Alternate Protocol 2: Nonurea Peptide Separations with Tris Buffers

  • Separating and stacking gel solutions (Table 3.0.7)
  • 2× Tris⋅Cl/SDS, pH 8.8 (dilute 4× Tris⋅Cl/SDS, pH 8.8; Table 3.0.1)
  • 2× SDS electrophoresis buffer (see recipe)

Alternate Protocol 3: Continuous SDS‐PAGE

  • Separating gel solution (Table 3.0.8)
  • 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 3.0.9)
  • 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 3.0.10)
  • 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 3.0.11)
  • 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

   Adams, L. and Gallagher, S.R. 2004. Two‐dimension 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. 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. Curr. Protoc. Protein Sci. 00:10.2.1‐10.2.8.
   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.
   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. (ed.) 2002. Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed. Oxford University Press, New York.
  An excellent book describing gel electrophoresis of proteins.
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