Staining Proteins in Gels

Joachim Sasse1, Sean R. Gallagher2

1 Shriners Hospital for Crippled Children, Tampa, Florida, 2 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.9
DOI:  10.1002/0471142735.im0809s58
Online Posting Date:  February, 2004
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Abstract

This unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS‐polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

     
 
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Table of Contents

  • Detection of Proteins
  • Staining Proteins in Gels
  • Basic Protocol 1: Coomassie Blue Staining
  • Alternate Protocol 1: Rapid Coomassie Blue Staining
  • Basic Protocol 2: Silver Staining
  • Alternate Protocol 2: Nonammoniacal Silver Staining
  • Alternate Protocol 3: Rapid Silver Staining
  • Support Protocol 1: Gel Photography of Coomassie‐ or Silver‐Stained Gels
  • Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red
  • Alternate Protocol 4: Fluorescent Staining on Nondenaturing Gels Using SYPRO Orange or Red
  • Basic Protocol 4: Fluorescent Protein Stain Using SYPRO Ruby for 2‐D Gel Analysis
  • Support Protocol 2: Photography of Fluorescently Stained Gels
  • Alternate Protocol 5: Fluorescent Phosphoprotein Gel Staining for Selectively Staining Phosphoproteins in Polyacrylamide Gels
  • Support Protocol 3: Imaging and Documenting the Phosphoprotein‐Stained Gel
  • Support Protocol 4: Selective Detection of Phosphotyrosine Residues
  • Alternate Protocol 6: Fluorescent Staining for Differentially Staining Glycosylated and Nonglycosylated Proteins in the Same Gel
  • Support Protocol 5: Viewing and Photographing the Glycoprotein‐Stained Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Coomassie Blue Staining

  Materials
  • Polyacrylamide gel (units 8.4 & 8.5)
  • 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
  • Whatman 3MM filter paper (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: Silver Staining

  Materials
  • Polyacrylamide gel (units 8.4 & 8.5)
  • Fixing solution for Coomassie blue and silver staining (see recipe)
  • Methanol/acetic acid destaining solution (see recipe)
  • 10% (v/v) glutaraldehyde (freshly prepared from 50% stock; Kodak)
  • Silver nitrate solution (see recipe)
  • Developing solution (see recipe)
  • Kodak Rapid Fix Solution A
  • Whatman 3MM filter paper (optional)
  • Gel dryer (optional)
NOTE: Wear gloves at all times to avoid fingerprint contamination.

Alternate Protocol 2: Nonammoniacal Silver Staining

  • 5 µg/ml dithiothreitol (DTT; appendix 2A)
  • 0.1% (w/v) silver nitrate solution (store in brown bottle at room temperature up to ∼1 month)
  • Carbonate developing solution (see recipe)
  • 2.3 M citric acid
  • 0.03% (w/v) sodium carbonate (optional)

Alternate Protocol 3: 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

Support Protocol 1: Gel Photography of Coomassie‐ or Silver‐Stained Gels

  Materials
  • SYPRO Orange or Red fluorescent staining solution (see recipe)
  • 7.5% (v/v) acetic acid
  • 0.1% (v/v) Tween‐20
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 8.4)

Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red

  Materials
  • 1‐D or 2‐D polyacrylamide or IEF gels (units 8.4 & 8.5)
  • Fixing solution for SYPRO Ruby staining of 2‐D polyacrylamide gels (see recipe)
  • Fixing solution for IEF gels: 40% (v/v) methanol/10% (w/v) trichloroacetic acid in H 2O
  • SYPRO Ruby protein gel stain (Molecular Probes; see recipe)
  • 10% (v/v) methanol (or ethanol)/7% (v/v) acetic acid
  • 2% (v/v) glycerol
  • Plastic staining dishes of appropriate size for gels: polypropylene (e.g., Rubbermaid Servin' Savers) or PVC photographic staining trays (e.g., Photoquip Cesco‐Lite 8‐in. × 10‐in., for large‐format 2‐D gels)
  • Orbital shaker
  • Additional reagents and equipment for photography of fluorescently stained gels (see protocol 10)

Alternate Protocol 4: Fluorescent Staining on Nondenaturing Gels Using SYPRO Orange or Red

  Materials
  • Protein‐containing sample of interest
  • Methanol, spectroscopy grade
  • Chloroform, spectroscopy grade
  • Appropriate 1× sample buffer for electrophoresis (units 8.4 & 8.5)
  • Phosphoprotein standards (PeppermintStick Phosphoprotein Molecular Weight Standards, Molecular Probes; also see Table 8.9.1)
    Table 8.9.1   MaterialsExamples of Commercially Available Phosphorylated and Nonphosphorylated Proteins for Use as Controls

    Protein Mol. wt. (Da) Number of phosphate residues Lower limit of detection
    Riboflavin‐binding protein 29,200 8 1–3 ng
    α‐Casein 23,600 8 1–2 ng
    β‐Casein 24,500 5 1–2 ng
    Ovalbumin 45,000 2 4–8 ng
    Pepsin 35,500 1 8–16 ng
    Carbonic anhydrase 30,000 0 Not applicable
    Bovine serum albumin (BSA) 66,000 0 Not applicable

  • Fixing solution for phosphoprotein gels (see recipe)
  • Pro‐Q Diamond phosphoprotein gel stain (Molecular Probes; see recipe)
  • Phosphoprotein gel destain solution (see recipe, or purchase from Molecular probes)
  • Polystyrene staining dish (e.g., a weighing dish)
  • Orbital shaker
  • Additional reagents and equipment for polyacrylamide gel electrophoresis (units 8.4 & 8.5), SYPRO Ruby protein gel staining (see 8.9), Coomassie blue stain (see 8.9), silver staining (see 8.9), and imaging and documenting phosphoprotein‐stained gels

Basic Protocol 4: Fluorescent Protein Stain Using SYPRO Ruby for 2‐D Gel Analysis

  • Gel for phosphoprotein fluorescent staining (either before fixation/staining or after fixation/staining/destaining; see protocol 11)
  • Barium hydroxide octahydrate
  • Argon source
  • Glacial acetic acid
  • 50°C shaking water bath

Support Protocol 2: Photography of Fluorescently Stained Gels

  Materials
  • Protein‐containing sample of interest
  • 8‐cm × 10‐cm SDS‐ polyacrylamide minigel (unit 8.4)
  • Sample buffer (unit 8.4)
  • Pro‐Q Emerald glycoprotein gel staining kit (Molecular Probes) including:
    • Pro‐Q Emerald 300 staining reagent (component A), 50× concentrate in DMF (store at −20°C up to 6 months, protected from light)
    • Pro‐Q Emerald 300 staining buffer (component B; store at room temperature up to 6 months)
    • Oxidizing reagent (component C): 2.5 g of periodic acid (add 250 ml of 3% v/v acetic acid and store at room temperature up to 6 months)
    • CandyCane glycoprotein molecular weight standards (store at −20°C up to 6 months)
  • Fixing solution: 50% (v/v) methanol in H 2O
  • Wash solution: 3% (v/v) glacial acetic acid in H 2O
  • Polystyrene staining dish (e.g., large weighing dish)
  • Orbital shaker
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4), viewing and documenting glycoprotein‐stained gels (see protocol 15), and SYPRO Ruby staining (see protocol 9)
NOTE: The following procedure is optimized for staining 0.5‐ to 0.75‐mm thick, 8‐cm × 10‐cm minigels. Large 2‐D gels (20 cm × 20 cm) require much larger volumes and longer fixation and staining times, as indicated in the annotations to the respective steps.
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, N.L. 1988. Two Dimensional Electrophoresis Operation of the ISO‐DALT (R) System; Large Scale Biology Press, Washington, D.C.
   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.
   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.
   Saase, J. 1991. Detection of proteins on blot transfer membranes. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.7.1‐10.7.3. John Wiley & Sons, New York.
   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.
   Switzer, R.C., Merril, C.R., and Shifrin, S. 1979. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal. Biochem. 98:231‐237.
   Wilson, C.M. 1983. Staining of proteins on gels: Comparison of dyes and procedures. Methods Enzymol. 91:236‐247.
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