Staining Proteins in Gels

Sean R. Gallagher1, Joachim Sasse2

1 UVP, LLC, Upland, California, 2 Biomedtech Laboratories, Inc., Tampa, Florida
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 7.4
DOI:  10.1002/9780470089941.et0704s06
Online Posting Date:  August, 2012
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Abstract

This unit describes protocols for detecting proteins in SDS‐polyacrylamide gels. It describes methods for Coomassie blue and silver staining, as well as the fluorescent stains SYPRO Orange and Red. Staining with Coomassie blue is easier and more rapid; however, silver staining methods are considerably more sensitive and can thus be used to detect smaller amounts of protein. Alternative rapid staining procedures are provided for each method. Fluorescent staining is a popular alternative to the traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Curr. Protoc. Essential Lab. Tech. 6:7.4.1‐7.4.14. © 2012 by John Wiley & Sons, Inc.

Keywords: silver; Coomassie; stain; protein; fluorescence; electrophoresis; SYPRO

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Protocols
  • Basic Protocol 1: Coomassie Blue Staining
  • Alternate Protocol 1: Rapid Coomassie Blue Staining
  • Basic Protocol 2: Nonammoniacal Silver Staining
  • Alternate Protocol 2: Rapid Silver Staining
  • Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red
  • Support Protocol 1: Photography of SYPRO Orange or Red Fluorescently Stained Gels
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Coomassie Blue Staining

  Materials
  • Polyacrylamide gel with separated proteins (see unit 7.3)
  • Fixing solution for Coomassie blue and silver staining (see recipe)
  • Coomassie blue staining solution (see recipe)
  • Methanol/acetic acid destaining solution (see recipe)
  • 7% (v/v) aqueous acetic acid
  • Plastic container (slightly larger than the gel)
  • Orbital shaker or rocking platform
  • Heat‐sealable plastic bags
  • Imaging system with Coomassie blue filter (see Table 7.4.1)
  • Camera (optional)
  • Whatman No. 3 MM filter paper (optional; Whatman #3030‐6189; 0.34 mm thickness)
  • Plastic wrap (optional)
  • Gel dryer (optional)

Alternate Protocol 1: Rapid Coomassie Blue Staining

  • Isopropanol fixing solution (see recipe)
  • Rapid Coomassie blue staining solution (see recipe)
  • 10% (v/v) acetic acid

Basic Protocol 2: Nonammoniacal Silver Staining

  Materials
  • Polyacrylamide gel with separated proteins (unit 7.3)
  • Fixing solution for Coomassie blue and silver staining (see recipe)
  • Methanol/acetic acid destaining solution (see recipe)
  • 10% (v/v) glutaraldehyde: prepare fresh from 50% stock (Kodak)
  • 5 µg/ml dithiothreitol (DTT; unit 3.3)
  • 0.1% (w/v) silver nitrate solution: prepare fresh before use
  • Carbonate developing solution (see recipe)
  • 2.3 M citric acid
  • 0.03% (w/v) sodium carbonate (optional)
  • Glass or polyethylene container (slightly bigger than the gel)
  • Orbital shaker or rocking platform
  • Digital imaging system with silver stain filter (see Table 7.4.1)
  • White light table for transillumination of the gel
  • Camera (optional)
  • Gel dryer (optional)
  • Heat‐sealable plastic bags (optional)
NOTE: Wear gloves at all times to avoid fingerprint contamination.

Alternate Protocol 2: Rapid Silver Staining

  • Formaldehyde fixing solution (see recipe)
  • 0.2 g/liter sodium thiosulfate (Na 2S 2O 3)
  • Thiosulfate developing solution (see recipe)
  • Drying solution (see recipe)
  • Dialysis membrane soaked in 50% methanol
  • Glass plates

Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red

  Materials
  • Protein sample for analysis
  • SYPRO Orange or Red fluorescent staining solution (see recipe)
  • 7.5% (v/v) acetic acid
  • 0.1% (v/v) Tween 20
  • Ultraviolet Transilluminator (UVP, Bio‐Rad)
  • Ultraviolet to blue light converter plate for use with UV transilluminator (UVP, Bio‐Rad)
  • Blue light transilluminator (UVP, Life Technologies, Bio‐Rad)
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 7.3)
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Figures

Videos

Literature Cited

Literature Cited
   Bloom, H., Beier, H., and Gross, H.S. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93‐99.
   Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggen, G.M., Carnerolla, B., Orecchia, P., Zardi, L., and Righetti, P.G. 2004. Blue silver: A very sensitive colloidal Coomassie G‐250 staining for proteome analysis. Electrophoresis 25:1327‐1333.
   Fazekas de St. Groth, S., Webster, R.G., and Datyner, A. 1963. Two new staining procedures for quantitative estimation of proteins on electrophoretic strips. Biochim. Biophys. Acta 71:337‐391.
   Merril, C.R. 1990. Silver staining of proteins and DNA. Nature 343:779‐780.
   Merril, C.R., Goldman, D., and Van Keuren, M.L. 1984. Gel protein stains: Silver stain. Methods Enzymol. 104:441‐447.
   Morrissey, J.H. 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307‐310.
   Oakley, B.R., Kirsch, D.R., and Morris, N.R. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105:361‐363.
   Rabilloud, T. 1990. Mechanisms of protein silver staining in polyacrylamide gels: A 10‐year synthesis. Electrophoresis 11:785‐794.
   Steinberg, T.H., Haugland, R.P., and Singer, V.L. 1996a. Applications of SYPRO orange and SYPRO red protein gel stains. Anal. Biochem. 239:238‐245.
   Steinberg, T.H., Jones, L.J., Haugland, R.P., and Singer, V.L. 1996b. SYPRO orange and SYPRO red protein gel stains: One‐step fluorescent staining of denaturing gels for detection of nanogram levels of protein. Anal. Biochem. 239:223‐237.
   Steinberg, T.H., White, H.M., and Singer, V.L. 1997. Optimal filter combinations for photographing SYPRO orange or SYPRO red dye‐stained gels. Anal. Biochem. 248:168‐172.
   Westermeier, R. 2006. Sensitive, quantitative, and fast modifications for Coomassie blue staining of polyacrylamide gels. Proteomics 6:61‐64.
   Wilson, C.M. 1983. Staining of proteins on gels: Comparison of dyes and procedures. Methods Enzymol. 91:236‐247.
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