Protein Analysis by SDS‐PAGE and Detection by Coomassie Blue or Silver Staining

Sean Gallagher (electrophoresis, staining)1, Joachim Sasse (staining)2

1 Motorola Phoenix Applied Research Center, Tempe, Arizona, 2 Shriners Hospital, Tampa, Florida
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Appendix 3B
DOI:  10.1002/0471141755.pha03bs02
Online Posting Date:  May, 2001
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Abstract

Electrophoresis is used to separate complex mixtures of proteins, 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, the combination of gel pore size and protein charge, size, and shape determines the migration rate of the protein. This unit first presents an informative section about safe use of electricity and electrophoresis. Protocols are then provided for one‚Äźdimensional gel electrophoresis under denaturing conditions and subsequent staining with either Coomassie blue or silver staining.Electrophoresis is used to separate complex mixtures of proteins, to investigate subunit compositions and to verify homogeneit

     
 
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Table of Contents

  • Electricity and Electrophoresis
  • Basic Protocol 1: Denaturing (SDS) Discontinuous Gel Electrophoresis: Laemmli Gel Method
  • Staining Proteins in Gels
  • Support Protocol 1: Coomassie Blue Staining
  • Support Protocol 2: Silver Staining
  • Reagents and Solutions
  • 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; see recipe)
  • Protein sample to be analyzed
  • 2× and 1× SDS sample buffer (see recipe)
  • Protein‐molecular‐weight standards mixture (e.g., Bio‐Rad; see Table 3.0.2)
  • recipe6× SDS sample buffer (optional)
  • 1× SDS electrophoresis buffer (see recipe)
    Table 0.b.2   Materials   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis e   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis   Recipes for Polyacrylamide Separating and Stacking Gels a   Recipes for Polyacrylamide Separating and Stacking Gels

    Protein Molecular weight
    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
    SEPARATING GEL
    Stock solution b Final acrylamide concentration in separating gel (%) c
    5 6 7 7.5 8 9 10 12 13 15
    30% acrylamide/0.8% 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 6.8 (see recipe), and H 2O in the amounts specified above. Degas under vacuum about 5 min. Add the 10% ammonium persulfate solution and TEMED (N,N,N′,N′‐tetramethylethylenediamine). Swirl gently to mix. Use immediately.
    STACKING GEL(3.9% 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 recipe), 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. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

     eProtein standards are commercially available in kits (e.g., Hoefer Pharmacia Biotech, Life Technologies, Bio‐Rad, or Sigma).
  • Electrophoresis apparatus with clamps, glass plates, casting stand, and buffer chambers (Bio‐Rad, Hoefer Pharmacia Biotech)
  • 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
  • 1.5‐ml screw‐cap microcentrifuge tube
  • 25‐ or 100‐µl syringe with flat‐tipped needle
  • Constant‐current power supply
    Table 0.b.1   Materials   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis e   Molecular Weights of Protein Standards for Polyacrylamide Gel Electrophoresis   Recipes for Polyacrylamide Separating and Stacking Gels a   Recipes for Polyacrylamide Separating and Stacking Gels

    Protein Molecular weight
    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
    SEPARATING GEL
    Stock solution b Final acrylamide concentration in separating gel (%) c
    5 6 7 7.5 8 9 10 12 13 15
    30% acrylamide/0.8% 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 6.8 (see recipe), and H 2O in the amounts specified above. Degas under vacuum about 5 min. Add the 10% ammonium persulfate solution and TEMED (N,N,N′,N′‐tetramethylethylenediamine). Swirl gently to mix. Use immediately.
    STACKING GEL(3.9% 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 recipe), 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. Failure to form a firm gel usually indicates a problem with the persulfate, TEMED, or both.

     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 (1970).
     bAll reagents and solutions used in the protocol must be prepared with water purified using a Milli‐Q‐filter (Millipore) or equivalent. See for recipes.
     cUnits of numbers in table body are milliliters. The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated. See annotation to step .
     dBest to prepare fresh.

Support Protocol 1: Coomassie Blue Staining

  Materials
  • Polyacrylamide gel (see protocol 1)
  • Fixing solution (see recipe)
  • Coomassie staining solution (see recipe)
  • Destaining solution (see recipe)
  • 7% (v/v) aqueous acetic acid
  • Cellophane membrane backing sheets (optional; Hoefer Pharmacia Biotech or Bio‐Rad)
  • Appropriate photographic filter and film (optional): yellow‐orange filter (e.g., Wratten no. 8 or no. 9 filter series A) with fine‐grained panchromatic halftone film (e.g., Kodak T‐Max 100)
  • Whatman 3MM filter paper (optional)
NOTE: Deionized, distilled water should be used throughout this protocol.

Support Protocol 2: Silver Staining

  Materials
  • Fixing and destaining solutions (see recipe)
  • 10% (v/v) glutaraldehyde (freshly prepared from 50% stock; Eastman Kodak)
  • Silver nitrate solution (ammoniacal; see recipe)
  • Developing solution (see recipe)
  • Kodak Rapid Fix Solution A
  • Appropriate photographic filter and film (optional): blue‐green filter (e.g., Wratten no. 58) with Kodak T‐Max 100 or equivalent
NOTE: Wear gloves at all times to avoid fingerprint contamination.
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Figures

Videos

Literature Cited

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