Protein Detection in Gels Using Fixation

Lynn A. Echan1, David W. Speicher1

1 The Wistar Institute, Philadelphia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.5
DOI:  10.1002/0471140864.ps1005s29
Online Posting Date:  November, 2002
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Abstract

This unit has been recently updated to include information on preformulated gel stains as well as new protocols for Sypro Ruby and silver staining, and gel imaging methodology. Other previously published protocols are also provided for both rapid and acid‐based Coomassie blue staining, and alternate methods for silver staining (i.e., nonammoniacal silver staining, rapid silver staining, and an enhanced‐background, two‐stage method).

     
 
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Table of Contents

  • Basic Protocol 1: Coomassie Blue R‐250 Staining
  • Alternate Protocol 1: Rapid Coomassie Blue G‐250 Staining
  • Alternate Protocol 2: Acid‐Based Coomassie Blue G‐250 Staining
  • Basic Protocol 2: SYPRO Ruby Staining
  • Basic Protocol 3: Silver Staining
  • Alternate Protocol 3: Nonammoniacal Silver Staining
  • Alternate Protocol 4: Rapid Silver Staining
  • Alternate Protocol 5: Enhanced‐Background (Two‐Stage) Rapid Silver Staining
  • Support Protocol 1: Gel Imaging
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Coomassie Blue R‐250 Staining

  Materials
  • recipeCoomassie blue R‐250 staining solution (see recipe)
  • Polyacrylamide gel containing protein of interest (units 10.1 10.4)
  • Destaining solution: 15% methanol/10% acetic acid (v/v; store up to 1 month at room temperature)
  • 7% (v/v) acetic acid (optional)
  • Plastic or glass container with tight‐fitting lid of size appropriate for gel
  • Platform shaker
  • Protein‐based destaining material (optional): white 100% wool yarn, cellulose sponge, or Whatman 3MM filter paper
  • Resealable plastic bags
  • Gel dryer (optional)
NOTE: Only high‐purity water such as Milli‐Q‐purified water or equivalent should be used throughout the protocol, unless otherwise specified.

Alternate Protocol 1: Rapid Coomassie Blue G‐250 Staining

  • Isopropanol fixing solution: 25% isopropanol/10% acetic acid (v/v; store several months at room temperature)
  • Rapid Coomassie blue G‐250 staining solution: 10% (v/v) acetic acid/0.006% (w/v) Coomassie brilliant blue G‐250 (Bio‐Rad; store several months at room temperature)
  • 10% (v/v) acetic acid

Alternate Protocol 2: Acid‐Based Coomassie Blue G‐250 Staining

  • recipeAcid‐based Coomassie blue G‐250 staining solution (see recipe)

Basic Protocol 2: SYPRO Ruby Staining

  Materials
  • SDS 1‐ or 2‐D polyacrylamide gels containing proteins of interest (units 10.1 10.4)
  • Fixing solution: 40% (v/v) methanol/10% (v/v) acetic acid in H 2O
  • SYPRO Ruby protein gel stain (Molecular Probes)
  • Destain/wash solution: 2% (v/v) acetic acid
  • 2% (w/v) glycerol
  • Plastic container with tight‐fitting lid, of size appropriate for gel
  • Platform shaker
  • Additional reagents and equipment for acquiring gel images (see protocol 9)

Basic Protocol 3: Silver Staining

  Materials
  • recipePolyacrylamide gel containing proteins of interest (see units 10.1 10.4; also see 10.5)
  • Fixing solution A: 40% (v/v) ethanol/10% (v/v) acetic acid in H 2O
  • Fixing solution B: 5% (v/v) ethanol/5% (v/v) acetic acid in H 2O
  • 0.5 M sodium acetate containing 1% (v/v) glutaraldehyde
  • 0.05% (w/v) 2,7‐napthalene disulfonic acid
  • recipeAmmoniacal silver nitrate solution (see recipe)
  • recipeCitric acid developing solution (see recipe)
  • Stop solution: 5% (w/v) Tris base containing 2% (v/v) acetic acid
  • Glass or plastic container with tight‐fitting lid, of size appropriate for gel
  • Platform shaker
  • Resealable plastic bags
  • Additional reagents and equipment for acquiring gel images (see protocol 9)
CAUTION: Ammoniacal silver nitrate solution becomes explosive on drying. Discard immediately after use by adding 1 M HCl to precipitate the silver as silver chloride and dispose of as required by local regulations.

Alternate Protocol 3: Nonammoniacal Silver Staining

  Materials
  • Fixing solution A: 50% (v/v) methanol/10% (v/v) acetic acid (store up to 1 month at room temperature)
  • Fixing solution B: 5% (v/v) methanol/7% (v/v) acetic acid (store up to 1 month at room temperature)
  • 10% (w/v) glutaraldehyde
  • 5 µg/ml dithiothreitol (DTT; make fresh daily)
  • 0.1% (w/v) silver nitrate
  • recipeCarbonate developing solution (see recipe)
  • 2.3 M citric acid
  • 0.03% (w/v) sodium carbonate
  • Plastic or glass container with tight‐fitting lid, of size appropriate for gel
  • Platform shaker

Alternate Protocol 4: Rapid Silver Staining

  Materials
  • 50% methanol/12% acetic acid (v/v)
  • 95% ethanol (optional)
  • recipeFormaldehyde fixing solution (see recipe)
  • 0.02% (w/v) sodium thiosulfate
  • 0.1% (w/v) silver nitrate
  • recipeThiosulfate developing solution (see recipe)
  • 50% methanol
  • Plastic or glass container with tight‐fitting lid, of size appropriate for gel
  • Platform shaker

Alternate Protocol 5: Enhanced‐Background (Two‐Stage) Rapid Silver Staining

  Materials
  • Rapid fixative 1: 40% methanol/10% acetic acid (v/v; store 1 month at room temperature)
  • Rapid fixative 2: 10% methanol/5% acetic acid (v/v; store 1 month at room temperature)
  • recipe1× oxidizer (see recipe)
  • 0.17% (w/v) silver nitrate
  • recipeThiosulfate developing solution (see recipe)
  • 5% (w/v) acetic acid
  • recipeFarmers reducer (see recipe)
  • Plastic or glass container with tight‐fitting lid, of size appropriate for gel
  • Platform shaker
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Figures

Videos

Literature Cited

Literature Cited
   Blakesley, R.W. and Boezi, J.A. 1977. A new staining technique for protein in polyacrylamide gels using Coomassie Brilliant Blue G‐250. Anal. Biochem. 82:580‐582.
   Blum, H., Beier, H., and Goss, H.J. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93‐99.
   Goforth, S. 2001. Exposing gel documentation systems. The Scientist. 15:28,
   Gorg, A. 1985. Horizontal SDS electrophoresis in ultrathin pore gradient gels for the analysis of urinary proteins. Sci. Tools 32:5‐9.
   Miller, M.D., Acey, R.A., Lee, L.Y.T., and Edwards, A.J. 2001. Digital imaging considerations for gel electrophoresis analysis systems. Electrophoresis 22:791‐800.
   Miura, K. 2001. Imaging and detection technologies for image analysis in electrophoresis. Electrophoresis 22:801‐813.
   Morrissey, J.H. 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307‐310.
   Patton, W.F. 2000. A thousand points of light: The application of fluorescence detection technologies to two‐dimensional gel electrophoresis and proteins. Electrophoresis. 21:1123‐1144.
   Weber, K., Pringle, J.R., and Osborn, M. 1972. Measurement of molecular weights by electrophoresis on SDS‐acrylamide gel. Methods Enzymol. 26:3‐27.
   Yan, J.X., Harry, R.A., Spibey, C., and Dunn, M.J. 2000. Postelectrophoretic staining of proteins separated by two‐dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 21:3675‐3665.
Key References
   Miller et al., 2001. See above.
  Overview of gel‐imaging devices.
   Patton, 2000. See above.
  Describes fluorescent stains.
   Rabilloud, T. 1992. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13:429‐439.
  Compares many different silver staining methods.
   Wilson, C. 1983. Staining of proteins on gels: Comparison of dyes and procedures. Methods Enzymol. 91:236‐247.
  Reviews different Coomassie blue staining procedures.
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