One‐Dimensional SDS Gel Electrophoresis of Proteins

Sean R. Gallagher1

1 UVP, LLC, Upland, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.2A
DOI:  10.1002/0471142727.mb1002as97
Online Posting Date:  January, 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. Mol. Biol. 97:10.2A.1‐10.2A.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 10.2.1)
  • H 2O‐saturated isobutyl alcohol
  • 1× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.2.1)
  • Protein sample, on ice
  • 2× and 1× SDS sample buffer (see recipe)
  • Protein molecular weight standards (Tables 10.2.2 and 10.2.3)
  • 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 10.2.5)
  • 2× tricine sample buffer (see recipe)
  • Peptide molecular weight standards (Table 10.2.6)
  • 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 0.2.5   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Tricine Peptide Separating and Stacking Gels l   Recipes for Tricine Peptide Separating and Stacking Gels   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis

    SEPARATING AND STACKING GELS
    Stock solution m 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 n 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 10.2.1), 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

     lThe 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 ( ).
     mAll reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     nBest to prepare fresh. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.
    Table 0.2.6   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Tricine Peptide Separating and Stacking Gels l   Recipes for Tricine Peptide Separating and Stacking Gels   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis   Molecular Weights of Peptide Standards for Polyacrylamide Gel Electrophoresis

    SEPARATING AND STACKING GELS
    Stock solution m 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 n 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 10.2.1), 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

     Peptide 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 10.2.7)
  • 2× Tris·Cl/SDS, pH 8.8 (dilute 4× Tris·Cl/SDS, pH 8.8; Table 10.2.1)
  • 2× SDS electrophoresis buffer (see recipe)
    Table 0.2.7   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Modified Laemmli Peptide Separating and Stacking Gels   Recipes for Modified Laemmli Peptide Separating and Stacking Gels

    SEPARATING AND STACKING GELS
    Stock solution Separating gel Stacking gel
    30% acrylamide/0.8% 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 0.15 ml 50 µl
    H 2O 1.00 ml 3.00 ml
    10% (w/v) ammonium persulfate 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 10.2.1), 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.

     The 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. ( ).
     All reagents and solutions used in the protocol must be prepared with Milli‐Q‐purified water or equivalent.
     Best 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 10.2.8)
  • 2× and 1× phosphate/SDS sample buffer (see recipe)
  • 1× phosphate/SDS electrophoresis buffer (see recipe)
    Table 0.2.8   Additional Materials (also see protocol 1)   Additional Materials   Recipes for Separating Gels for Continuous SDS‐PAGE   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% acrylamide/0.8% 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 10.2.1), 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% 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 up to 3 months at 4°C.

     The 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
  • 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 10.2.9)
  • 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 0.2.9   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% acrylamide/0.8% 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 10.2.1), 4× Tris·Cl/SDS, pH 8.8 (Table 10.2.1), 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 10.2.1), 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 10.2.10)
  • 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 0.2.0   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 10.2.1 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 10.2.11)
  • 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 0.2.1   Additional Materials (also see protocol 7)   Additional MaterialsLight and Heavy Acrylamide Gel Solutions for Casting Multiple Gradient Gels

    Acrylamide concentration in light gel solution (%) a,b
    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 (%) a,b
    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 10.2.1 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

  •   FigureFigure 10.2.1 Series and parallel connections of gel tanks to power supply.
  •   FigureFigure 10.2.2 Gradient gel setup. A peristaltic pump, though not required, will provide better control.
  •   FigureFigure 10.2.3 Setup for casting multiple gradient gels. Casting multiple gradient gels requires a peristaltic pump and a multiple gel caster. Gel solution is introduced through the bottom of the multiple caster.
  •   FigureFigure 10.2.4 Minigel sandwiches positioned in the multiple gel caster. Extra glass or acrylic plates or polycarbonate sheets are used to fill any free space in the caster and to ensure that the gel sandwiches are held firmly in place.
  •   FigureFigure 10.2.5 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 lenxgths. 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.
  •   FigureFigure 10.2.6 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 ( UNIT), 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.
  •   FigureFigure 10.2.7 Structures of acrylamide and bisacrylamide and the associated reaction producing the polyacrylamide matrix used for protein separation.
  •   FigureFigure 10.2.8 Structures of sodium dodecyl sulfate, dithiothreitol, and 2‐mercaptoethanol: used to break disulfide bonds in proteins so they are fully denatured.
  •   FigureFigure 10.2.9 Separation of membrane proteins by 5.1% to 20.5% T polyacrylamide gradient SDS‐PAGE. Approximately 30 µl of 1× SDS sample buffer containing 30 µg of Alaskan pea ( Pisum sativum) membrane proteins was loaded in wells of a 14 × 14–cm, 0.75‐mm‐thick gel. Standard proteins were included in the outside lanes. The gel was run at 4 mA for ≈15 hr.
  •   FigureFigure 10.2.10 A typical version of a form used for recording gel data.

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.
   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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