Staining Proteins in Gels

Esteban C. Dell'Angelica1, Juan S. Bonifacino1

1 National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.6
DOI:  10.1002/0471143030.cb0606s06
Online Posting Date:  May, 2001
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Abstract

Once proteins are separated by gel electrophoresis, staining can be used to visualize the proteins. This unit presents protocols for numerous staining methods. The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear background. This technique can also be applied to isoelectric focusing gels. A second, more sensitive but also more technically challenging method is silver staining. Here the proteins are seen as dark brown to black bands on a clear background. If the gel is incubated with SYPRO Ruby, a fluorescent compound that interacts specifically with proteins, the bands fluoresce when illuminated on a standard transilluminator. Finally, proteins can be reversibly stained with zinc, which precipitates the SDS from the gel leaving protein bands as clear spots against an opaque white background.

     
 
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Table of Contents

  • Basic Protocol 1: Staining Protein Gels with Coomassie Blue
  • Alternate Protocol 1: Staining Protein Gels with Coomassie Blue After Isoelectric Focusing
  • Basic Protocol 2: Staining Protein Gels with Silver
  • Basic Protocol 3: Fluorescence Detection of Proteins in Gels
  • Basic Protocol 4: Reversible Protein Staining with Zinc
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Staining Protein Gels with Coomassie Blue

  Materials
  • Polyacrylamide gel containing protein(s) of interest (see unit 6.1)
  • Coomassie blue staining solution (see recipe)
  • Destaining solution: 30% (v/v) methanol/10% (v/v) acetic acid in distilled water (store up to 4 months at room temperature)
  • Storage solution: 7% (v/v) acetic acid/5% (v/v) methanol in distilled water (store up to 4 months at room temperature)
  • Plastic container with lid (pipet tip containers are appropriate for staining mini‐gels)
  • Platform shaker (optional)
CAUTION: Glacial acetic acid and methanol are volatile and toxic. The destaining and storage solutions should be prepared in a chemical fume hood, and gloves should be worn throughout the staining procedure.NOTE: All steps should be performed at room temperature.

Alternate Protocol 1: Staining Protein Gels with Coomassie Blue After Isoelectric Focusing

  • Polyacrylamide isoelectric focusing gel with protein(s) of interest (e.g., units 6.4 & 15.4)
  • 20% (w/v) TCA solution in water
  • IEF Coomassie blue stock solution (see recipe)
  • 10% (w/v) CuSO 4 in distilled water (store for up to 4 months at room temperature)
CAUTION: TCA is extremely caustic. Protect eyes and wear gloves when preparing and handling TCA solutions.

Basic Protocol 2: Staining Protein Gels with Silver

  Materials
  • Polyacrylamide gel containing protein(s) of interest (see units 6.1, 6.4 & 6.5), either unfixed or fixed and stained with Coomassie blue (see 6.6 and 6.6)
  • Deionized water (HPLC grade or Milli‐Q)
  • 50% (v/v) ethanol in deionized water
  • Fixative solution (see recipe)
  • Thiosulfate solution (see recipe)
  • Silver nitrate solution (see recipe)
  • Developer solution (see recipe)
  • 50% (v/v) methanol/12% (v/v) acetic acid in deionized water
  • 50% (v/v) methanol in water
  • Plastic container with lid (pipet tip containers are appropriate for staining mini‐gels)
  • Clean plastic containers (at least six; lids of pipet tip containers are appropriate for staining mini‐gels)
  • Aluminum foil
  • Platform shaker (optional)
CAUTION: Glacial acetic acid and methanol are volatile and toxic; solutions containing these solvents should be prepared in a chemical fume hood and handled with care.NOTE: All solutions should be prepared in deionized water (Milli‐Q or HPLC grade). At all times wear gloves that have been rinsed extensively with distilled water. Handle the gel by using clean forceps having blunt tips or by touching the corners with clean, powder‐free gloves. All steps should be performed at room temperature.

Basic Protocol 3: Fluorescence Detection of Proteins in Gels

  Materials
  • Polyacrylamide gel containing protein(s) of interest (see unit 6.1, unit 6.4, or unit 6.5)
  • SYPRO Ruby protein gel stain (Molecular Probes)
  • Distilled water
  • Plastic container with lid (pipet tip containers are appropriate for staining mini‐gels)
  • Platform shaker (optional)
  • 300‐nm UV transilluminator
  • Photographic camera or CCD camera (optional)
CAUTION: The potential toxicity of the SYPRO Ruby dye, which comprises an organic component and ruthenium, has not been fully evaluated. Gloves should be worn throughout the staining procedure. For disposal, the stain solution should be poured through activated charcoal, and the dye adsorbed to activated charcoal destroyed in a chemical incinerator following local environmental regulations.NOTE: Protect SYPRO Ruby protein gel stain from light. If the plastic container with lid used for staining is transparent, cover it completely with aluminum foil.NOTE: All steps should be performed at room temperature.

Basic Protocol 4: Reversible Protein Staining with Zinc

  Materials
  • Polyacrylamide gel containing protein(s) of interest (see unit 6.1)
  • GelCode E‐Zinc Reversible Stain Kit (Pierce), containing:
  •  E‐Zinc Stain
  •  E‐Zinc Developer
  •  E‐Zinc Eraser
  • Distilled water
  • Plastic container with lid (pipet tip containers are appropriate for staining mini‐gels)
  • Platform shaker (optional)
NOTE: All steps should be performed at room temperature.
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Figures

Videos

Literature Cited

Literature Cited
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