One‐Dimensional SDS Gel Electrophoresis of Proteins

Sean R. Gallagher1

1 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.1
DOI:  10.1002/0471143030.cb0601s37
Online Posting Date:  December, 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 the 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. Curr. Protoc. Cell Biol. 37:6.1.1‐6.1.38. © 2007 by John Wiley & Sons, Inc.

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

     
 
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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 6.1.1)
  • H 2O‐saturated isobutyl alcohol
  • 1× Tris⋅Cl/SDS, pH 8.8 (dilute 4× Tris⋅Cl/SDS, pH 8.8; Table 6.1.1)
  • Protein sample, on ice
  • 2× and 1× SDS sample buffer (see recipe)
  • Protein molecular weight standards (Tables 6.1.2 and 6.1.3)
  • 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 above)
    Table 6.1.1   Materials   Recipes for Polyacrylamide Separating and Stacking Gels a   Recipes for Polyacrylamide Separating and Stacking Gels   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis e   Molecular Weights of Protein Standards for Polyacrylamide Gel ElectrophoresisProtein Standard Mixtures Available from Selected Suppliers

    SEPARATING GEL
    Final acrylamide concentration in separating gel (%) c
    Stock solution b 5 6 7 7.5 8 9 10 12 13 15
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 2.50 3.00 3.50 3.75 4.00 4.50 5.00 6.00 6.50 7.50
    4× Tris⋅Cl/SDS, pH 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 8.75 8.25 7.75 7.50 7.25 6.75 6.25 5.25 4.75 3.75
    10% (w/v) ammonium persulfate d 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
    Preparation of separating gel
    • In a 25‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution, 4× Tris⋅Cl/SDS, pH 8.8 (see reagents, below), and H 2O. Degas under vacuum ∼5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    STACKING GEL (3.9% w/v acrylamide)
    • In a 25‐ml side‐arm flask, mix 0.65 ml of 30% acrylamide/0.8% bisacrylamide, 1.25 ml of 4× Tris⋅Cl/SDS, pH 6.8 (see reagents, below), and 3.05 ml H 2O. Degas under vacuum 10 to 15 min. Add 25 µl of 10% ammonium persulfate and 5 µl TEMED. Swirl gently to mix. Use immediately.
    REAGENTS USED IN GELS
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide
    • Mix 30.0 g acrylamide and 0.8 g N,N′‐methylenebisacrylamide with H 2O in a total volume of 100 ml. Filter the solution through a 0.45‐µm filter and store at 4°C in the dark. The 2× crystallized grades of acrylamide and bisacrylamide are recommended. Discard after 30 days, as acrylamide gradually hydrolyzes to acrylic acid and ammonia.
    • CAUTION: Acrylamide monomer is neurotoxic. A mask should be worn when weighing acrylamide powder. Gloves should be worn while handling the solution, and the solution should not be pipetted by mouth.
    4× Tris⋅Cl/SDS, pH 6.8(0.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 6.05 g Tris base in 40 ml H 2O. Adjust to pH 6.8 with 1 N HCl. Add H 2O to 100 ml total volume. Filter the solution through a 0.45‐µm filter, add 0.4 g SDS, and store at 4°C up to 1 month.
    4× Tris⋅Cl/SDS, pH 8.8(1.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 91 g Tris base in 300 ml H 2O. Adjust to pH 8.8 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter, add 2 g SDS, and store at 4°C up to 1 month.
    Protein  Molecular weight (Da)
    Cytochrome c 11,700
    α‐Lactalbumin 14,200
    Lysozyme (hen egg white) 14,300
    Myoglobin (sperm whale) 16,800
    β‐Lactoglobulin 18,400
    Trypsin inhibitor (soybean) 20,100
    Trypsinogen, PMSF treated 24,000
    Carbonic anhydrase (bovine erythrocytes) 29,000
    Glyceraldehyde‐3‐phosphate dehydrogenase (rabbit muscle) 36,000
    Lactate dehydrogenase (porcine heart) 36,000
    Aldolase 40,000
    Ovalbumin 45,000
    Catalase 57,000
    Bovine serum albumin 66,000
    Phosphorylase b (rabbit muscle) 97,400
    β‐Galactosidase 116,000
    RNA polymerase, E. coli 160,000
    Myosin, heavy chain (rabbit muscle) 205,000
    Applications 1‐D 2‐D Im Pre Fluor Gly Phos Bio Tag IEF Nat
    Bio‐Rad X X X X X X
    CalBiochem X X
    Cell Signaling Technology X X X
    Favorgen X X
    GE Healthcare X X X X X X
    Invitrogen X X X X X X X X
    NEB X X
    Norgen Biotek X X
    Novagen X X X X
    PerkinElmer X X
    Pierce X X X X
    Promega X
    Qiagen X X X
    R & D Systems X X X
    Roche Applied Science X X
    Sigma‐Aldrich X X X X X X X X X X
    Upstate X X X
    USB X X X

     aThe recipes produce 15 ml of separating gel and 5 ml of stacking gel, which are adequate for a gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the SDS (denaturing) discontinuous buffer system of Laemmli ( ).
     bAll reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     cVolumes are in milliliters. The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated. See annotation to step 3, protocol 1.
     dBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.
     TEMED, N,N,N,N‐tetramethylethylenediamine.
    Table 6.1.2   Materials   Recipes for Polyacrylamide Separating and Stacking Gels a   Recipes for Polyacrylamide Separating and Stacking Gels   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis e   Molecular Weights of Protein Standards for Polyacrylamide Gel ElectrophoresisProtein Standard Mixtures Available from Selected Suppliers

    SEPARATING GEL
    Final acrylamide concentration in separating gel (%) c
    Stock solution b 5 6 7 7.5 8 9 10 12 13 15
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 2.50 3.00 3.50 3.75 4.00 4.50 5.00 6.00 6.50 7.50
    4× Tris⋅Cl/SDS, pH 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 8.75 8.25 7.75 7.50 7.25 6.75 6.25 5.25 4.75 3.75
    10% (w/v) ammonium persulfate d 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
    Preparation of separating gel
    • In a 25‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution, 4× Tris⋅Cl/SDS, pH 8.8 (see reagents, below), and H 2O. Degas under vacuum ∼5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    STACKING GEL (3.9% w/v acrylamide)
    • In a 25‐ml side‐arm flask, mix 0.65 ml of 30% acrylamide/0.8% bisacrylamide, 1.25 ml of 4× Tris⋅Cl/SDS, pH 6.8 (see reagents, below), and 3.05 ml H 2O. Degas under vacuum 10 to 15 min. Add 25 µl of 10% ammonium persulfate and 5 µl TEMED. Swirl gently to mix. Use immediately.
    REAGENTS USED IN GELS
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide
    • Mix 30.0 g acrylamide and 0.8 g N,N′‐methylenebisacrylamide with H 2O in a total volume of 100 ml. Filter the solution through a 0.45‐µm filter and store at 4°C in the dark. The 2× crystallized grades of acrylamide and bisacrylamide are recommended. Discard after 30 days, as acrylamide gradually hydrolyzes to acrylic acid and ammonia.
    • CAUTION: Acrylamide monomer is neurotoxic. A mask should be worn when weighing acrylamide powder. Gloves should be worn while handling the solution, and the solution should not be pipetted by mouth.
    4× Tris⋅Cl/SDS, pH 6.8(0.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 6.05 g Tris base in 40 ml H 2O. Adjust to pH 6.8 with 1 N HCl. Add H 2O to 100 ml total volume. Filter the solution through a 0.45‐µm filter, add 0.4 g SDS, and store at 4°C up to 1 month.
    4× Tris⋅Cl/SDS, pH 8.8(1.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 91 g Tris base in 300 ml H 2O. Adjust to pH 8.8 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter, add 2 g SDS, and store at 4°C up to 1 month.
    Protein  Molecular weight (Da)
    Cytochrome c 11,700
    α‐Lactalbumin 14,200
    Lysozyme (hen egg white) 14,300
    Myoglobin (sperm whale) 16,800
    β‐Lactoglobulin 18,400
    Trypsin inhibitor (soybean) 20,100
    Trypsinogen, PMSF treated 24,000
    Carbonic anhydrase (bovine erythrocytes) 29,000
    Glyceraldehyde‐3‐phosphate dehydrogenase (rabbit muscle) 36,000
    Lactate dehydrogenase (porcine heart) 36,000
    Aldolase 40,000
    Ovalbumin 45,000
    Catalase 57,000
    Bovine serum albumin 66,000
    Phosphorylase b (rabbit muscle) 97,400
    β‐Galactosidase 116,000
    RNA polymerase, E. coli 160,000
    Myosin, heavy chain (rabbit muscle) 205,000
    Applications 1‐D 2‐D Im Pre Fluor Gly Phos Bio Tag IEF Nat
    Bio‐Rad X X X X X X
    CalBiochem X X
    Cell Signaling Technology X X X
    Favorgen X X
    GE Healthcare X X X X X X
    Invitrogen X X X X X X X X
    NEB X X
    Norgen Biotek X X
    Novagen X X X X
    PerkinElmer X X
    Pierce X X X X
    Promega X
    Qiagen X X X
    R & D Systems X X X
    Roche Applied Science X X
    Sigma‐Aldrich X X X X X X X X X X
    Upstate X X X
    USB X X X

     eProtein standards are commercially available as prepared mixtures (see Table 6.1.3).
    Table 6.1.3   Materials   Recipes for Polyacrylamide Separating and Stacking Gels a   Recipes for Polyacrylamide Separating and Stacking Gels   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis e   Molecular Weights of Protein Standards for Polyacrylamide Gel ElectrophoresisProtein Standard Mixtures Available from Selected Suppliers

    SEPARATING GEL
    Final acrylamide concentration in separating gel (%) c
    Stock solution b 5 6 7 7.5 8 9 10 12 13 15
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 2.50 3.00 3.50 3.75 4.00 4.50 5.00 6.00 6.50 7.50
    4× Tris⋅Cl/SDS, pH 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 8.75 8.25 7.75 7.50 7.25 6.75 6.25 5.25 4.75 3.75
    10% (w/v) ammonium persulfate d 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
    Preparation of separating gel
    • In a 25‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution, 4× Tris⋅Cl/SDS, pH 8.8 (see reagents, below), and H 2O. Degas under vacuum ∼5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    STACKING GEL (3.9% w/v acrylamide)
    • In a 25‐ml side‐arm flask, mix 0.65 ml of 30% acrylamide/0.8% bisacrylamide, 1.25 ml of 4× Tris⋅Cl/SDS, pH 6.8 (see reagents, below), and 3.05 ml H 2O. Degas under vacuum 10 to 15 min. Add 25 µl of 10% ammonium persulfate and 5 µl TEMED. Swirl gently to mix. Use immediately.
    REAGENTS USED IN GELS
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide
    • Mix 30.0 g acrylamide and 0.8 g N,N′‐methylenebisacrylamide with H 2O in a total volume of 100 ml. Filter the solution through a 0.45‐µm filter and store at 4°C in the dark. The 2× crystallized grades of acrylamide and bisacrylamide are recommended. Discard after 30 days, as acrylamide gradually hydrolyzes to acrylic acid and ammonia.
    • CAUTION: Acrylamide monomer is neurotoxic. A mask should be worn when weighing acrylamide powder. Gloves should be worn while handling the solution, and the solution should not be pipetted by mouth.
    4× Tris⋅Cl/SDS, pH 6.8(0.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 6.05 g Tris base in 40 ml H 2O. Adjust to pH 6.8 with 1 N HCl. Add H 2O to 100 ml total volume. Filter the solution through a 0.45‐µm filter, add 0.4 g SDS, and store at 4°C up to 1 month.
    4× Tris⋅Cl/SDS, pH 8.8(1.5 M Tris⋅Cl containing 0.4% w/v SDS)
    • Dissolve 91 g Tris base in 300 ml H 2O. Adjust to pH 8.8 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter, add 2 g SDS, and store at 4°C up to 1 month.
    Protein  Molecular weight (Da)
    Cytochrome c 11,700
    α‐Lactalbumin 14,200
    Lysozyme (hen egg white) 14,300
    Myoglobin (sperm whale) 16,800
    β‐Lactoglobulin 18,400
    Trypsin inhibitor (soybean) 20,100
    Trypsinogen, PMSF treated 24,000
    Carbonic anhydrase (bovine erythrocytes) 29,000
    Glyceraldehyde‐3‐phosphate dehydrogenase (rabbit muscle) 36,000
    Lactate dehydrogenase (porcine heart) 36,000
    Aldolase 40,000
    Ovalbumin 45,000
    Catalase 57,000
    Bovine serum albumin 66,000
    Phosphorylase b (rabbit muscle) 97,400
    β‐Galactosidase 116,000
    RNA polymerase, E. coli 160,000
    Myosin, heavy chain (rabbit muscle) 205,000
    Applications 1‐D 2‐D Im Pre Fluor Gly Phos Bio Tag IEF Nat
    Bio‐Rad X X X X X X
    CalBiochem X X
    Cell Signaling Technology X X X
    Favorgen X X
    GE Healthcare X X X X X X
    Invitrogen X X X X X X X X
    NEB X X
    Norgen Biotek X X
    Novagen X X X X
    PerkinElmer X X
    Pierce X X X X
    Promega X
    Qiagen X X X
    R & D Systems X X X
    Roche Applied Science X X
    Sigma‐Aldrich X X X X X X X X X X
    Upstate X X X
    USB X X X

     Abbreviations: 1‐D, one‐dimensional gels; 2‐D, two‐dimensional gels; Im, immunoblotting; Pre, prestained; Fluor, fluorescent; Gly, glycoprotein; Phos, phosphoprotein; Bio, biotinylated; Tag, tagged; IEF, isoelectic focusing; Nat, native.
     2‐D standards are useful as independently characterized internal controls or reference standards for 2‐D SDS‐PAGE. Many investigators simply use an internally characterized test sample as a reference set.
     Prestained standards, while not as sharply delineated as unstained standards, can be used to monitor progress of the separation since the bands are visible through the gel cassette during electrophoresis. They are also useful for marking the position of a band after electroblotting to a nitrocellulose or PVDF membrane prior to immunoassay or analysis by mass spectrometry.

Alternate Protocol 1: Electrophoresis in Tris‐Tricine Buffer Systems

  • Separating and stacking gel solutions (Table 6.1.5)
  • 2× tricine sample buffer (see recipe)
  • Peptide molecular weight standards (Table 6.1.6)
  • 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
    Table 6.1.5   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% (w/v) acrylamide/0.8% (w/v) 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 25 µl
    TEMED 10 µl 5 µl
    Prepare separating and stacking gel solutions separately.
    • In a 50‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (Table 6.1.1), 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% w/v 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 6.1.6   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% (w/v) acrylamide/0.8% (w/v) 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 25 µl
    TEMED 10 µl 5 µl
    Prepare separating and stacking gel solutions separately.
    • In a 50‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (Table 6.1.1), 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% w/v 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 6.1.7)
  • 2× Tris⋅Cl/SDS, pH 8.8 (dilute 4× Tris⋅Cl/SDS, pH 8.8; Table 6.1.1)
  • 2× SDS electrophoresis buffer (see recipe)
    Table 6.1.7   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Modified Laemmli Peptide Separating and Stacking Gels k   Recipes for Modified Laemmli Peptide Separating and Stacking Gels

    SEPARATING AND STACKING GELS
    Stock solution l Separating gel Stacking gel
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 10.00 ml 0.65 ml
    8× Tris⋅Cl, pH 8.8 3.75 ml
    4× Tris⋅Cl, pH 6.8 1.25 ml
    10% (w/v) SDS m 0.15 ml 50 µl
    H 2O 1.00 ml 3.00 ml
    10% (w/v) ammonium persulfate m 50 µl 25 µl
    TEMED 10 µl 5 µl
    Prepare separating and stacking gel solutions separately.
    • In a 25‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (see Table 6.1.1), 8× Tris⋅Cl, pH 8.8, or 4× Tris⋅Cl, pH 6.8 (see reagents below), 10% SDS, and H 2O. Degas under vacuum 10 to 15 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    ADDITIONAL REAGENTS USED IN GELS
    4× Tris⋅Cl, pH 6.8(0.5 M Tris⋅Cl)
    • Dissolve 6.05 g Tris base in 40 ml H 2O. Adjust to pH 6.8 with 1 N HCl. Add H 2O to 100 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.
    8× Tris⋅Cl, pH 8.8(3.0 M Tris⋅Cl)
    • Dissolve 182 g Tris base in 300 ml H 2O. Adjust to pH 8.8 with 1 N HCl. Add H 2O to 500 ml total volume. Filter the solution through a 0.45‐µm filter and store up to 1 month at 4°C.

     kThe recipes produce 15 ml of separating gel and 5 ml of stacking gel, which are adequate for one gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the modified Laemmli peptide separation system of Okajima et al. ( ).
     lAll reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     mBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Alternate Protocol 3: Continuous SDS‐PAGE

  • Separating gel solution (Table 6.1.8)
  • 2× and 1× phosphate/SDS sample buffer (see recipe)
  • 1× phosphate/SDS electrophoresis buffer (see recipe)
    Table 6.1.8   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Separating Gels for Continuous SDS‐PAGE n   Recipes for Separating Gels for Continuous SDS‐PAGE

    SEPARATING GEL
    Final acrylamide concentration in the separating gel (%)
    Stock solution 5 6 7 8 9 10 11 12 13 14 15
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 2.5 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50
    4× phosphate/SDS, pH 7.2 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 8.75 8.25 7.75 7.25 6.75 6.25 5.75 5.25 4.75 4.25 3.75
    10% (w/v) ammonium persulfate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
    Preparation of separating gel
    • In a 25‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (see Table 6.1.1), 4× phosphate/SDS, pH 7.2, and H 2O. Degas under vacuum about 5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    ADDITIONAL REAGENTS USED IN GELS
    4× phosphate/SDS, pH 7.2(0.4 M sodium phosphate/0.4% w/v SDS)
    • Mix 46.8 g NaH 2PO 4⋅H 2O, 231.6 g Na 2HPO 4⋅7H 2O, and 12 g SDS in 3 liters H 2O. Store at 4°C for up to 3 months.

     nThe recipes produce 15 ml of separating gel, which is adequate for one gel of dimensions 0.75 mm × 14 cm × 14 cm. The recipes are based on the original continuous phosphate buffer system of Weber et al. ( ).
     All reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     Volumes are in milliliters. The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated. See protocol 1, annotation to step 3.
     Best to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Alternate Protocol 4: Casting and Running Ultrathin Gels

  • 95% (v/v) ethanol
  • GelBond (Lonza) 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 6.1.9)
  • 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
    Table 6.1.9   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Multiple Single‐Concentration Polyacrylamide Gels   Recipes for Multiple Single‐Concentration Polyacrylamide Gels

    SEPARATING GEL
    Stock solution Final acrylamide concentration in the separating gel (%)
    5 6 7 8 9 10 11 12 13 14 15
    30% (w/v) acrylamide/0.8% (w/v) bisacrylamide 52 62 72 83 93 103 114 124 134 145 155
    4× Tris⋅Cl/SDS, pH 8.8 78 78 78 78 78 78 78 78 78 78 78
    H 2O 181 171 160 150 140 129 119 109 98 88 78
    10% (w/v) ammonium persulfate 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
    TEMED 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21
    Preparation of separating gel
    • In a 500‐ml side‐arm flask, mix 30% acrylamide/0.8% bisacrylamide solution (see Table 6.1.1), 4× Tris⋅Cl/SDS, pH 8.8 (Table 6.1.1), and H 2O. Degas under vacuum ∼5 min. Add 10% ammonium persulfate and TEMED. Swirl gently to mix. Use immediately.
    STACKING GEL
    • In a 250‐ml side‐arm flask, mix 13.0 ml 30% acrylamide/0.8% bisacrylamide solution, 25 ml 4× Tris⋅Cl/SDS, pH 6.8 (Table 6.1.1), and 61 ml H 2O. Degas under vacuum ∼5 min. Add 0.25 ml 10% ammonium persulfate and 50 µl TEMED. Swirl gently to mix. Use immediately.

     The recipes produce about 300 ml of separating gel and 100 ml of stacking gel, which are adequate for ten gels of dimensions 1.5 mm × 14 cm × 14 cm, with extra solution should there be a leak or spill while casting the gels. For thinner spacers or fewer gels, calculate volumes using the equation in the annotation to step 4. The recipes are based on the SDS (denaturing) discontinuous buffer system of Laemmli ( ).
     All reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     Volumes in table body are in milliliters. The desired percentage of acrylamide in separating gel depends on the molecular size of the protein being separated. See protocol 1, annotation to step 3.
     Best to prepare fresh. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

Alternate Protocol 5: Separation of Proteins on Gradient Gels

  • Light and heavy acrylamide gel solutions (Table 6.1.10)
  • 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
    Table 6.1.0   Additional Materials (also see protocol 1)   Additional MaterialsLight and Heavy Acrylamide Gel Solutions for Gradient Gels

    Acrylamide concentration in light gel solution (%)a,b
    Stock solution 5 6 7 8 9 10 11 12 13 14
    30% acrylamide/0.8% bisacrylamide 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
    4× Tris⋅Cl/SDS, pH 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 8.75 8.25 7.75 7.25 6.75 6.25 5.75 5.25 4.75 4.25
    10% ammonium persulfate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005
    Acrylamide concentration in heavy gel solution (%)a,b
    Stock solution 10 11 12 13 14 15 16 17 18 19 20
    30% acrylamide/0.8% bisacrylamide 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
    4× Tris⋅Cl/SDS, pH 8.8 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75 3.75
    H 2O 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0
    Sucrose (g) 2.25 2.25 2.25 2.25 2.25 2.25 2.25 2.25 2.25 2.25 2.25
    10% ammonium persulfate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
    TEMED 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005

     To survey proteins ≥10 kDa, 5% to 20% gradient gels are recommended. To expand the range between 10 and 200 kDa, a 10% to 20% gradient gel is recommended.
     Volumes are in milliliters (sucrose is in grams). Keep light gel solution at room temperature prior to use (no longer than 1 hr). Keep heavy solution on ice.
     See Table 6.1.1 for preparation.
     Ammonium persulfate and TEMED are added directly to the gradient chambers immediately before the gel is poured. It is best to prepare ammonium persulfate fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

Support Protocol 2: Casting Multiple Gradient Gels

  • Plug solution (see recipe)
  • Light and heavy acrylamide gel solutions for multiple gradient gels (Table 6.1.11)
  • 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
    Table 6.1.1   Additional Materials (also see protocol 7)   Additional MaterialsLight and Heavy Acrylamide Gel Solutions for Casting Multiple Gradient Gels

    Acrylamide concentration in light gel solution (%)
    Stock solution 5 6 7 8 9 10 11 12 13 14
    30% acrylamide/0.8% bisacrylamide 28 33 39 44 50 55 61 66 72 77
    4× Tris·Cl/SDS, pH 8.8 41 41 41 41 41 41 41 41 41 41
    H 2O 96 91 85 80 74 69 63 58 52 47
    10% ammonium persulfate 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
    TEMED 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054
    Acrylamide concentration in heavy gel solution (%)
    Stock solution 10 11 12 13 14 15 16 17 18 19 20
    30% acrylamide/0.8% bisacrylamide 55 61 66 72 77 83 88 94 99 105 110
    4× Tris·Cl/SDS, pH 8.8 41 41 41 41 41 41 41 41 41 41 41
    H 2O 55 50 44 39 33 28 22 17 11 5.5 0
    Sucrose (g) 25 25 25 25 25 25 25 25 25 25 25
    10% ammonium persulfate 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
    TEMED 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054 0.054

     To survey proteins ≥10 kDa, 5% to 20% gradient gels are recommended. To expand the range between 10 and 200 kDa, a 10% to 20% gradient gel is recommended.
     Volumes are in milliliters (sucrose is in grams). Recipes produce ten 1.5‐mm‐thick gradient gels with 10 ml extra solution to account for losses in tubing. Keep light gel solution at room temperature prior to use (no longer than 1 hr). Keep heavy solution on ice.
     See Table 6.1.1 for preparation.
     Best to prepare fresh. Failure to form a firm gel usually indicates a problem with the ammonium persulfate, TEMED, or both.

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 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. 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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