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 Molecular Biology
Unit Number:  Unit 10.6
DOI:  10.1002/0471142727.mb1006s85
Online Posting Date:  January, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes protocols for detecting protein in a gel by Coomassie blue, silver, or fluorescent staining. As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Alternate protocols describe rapid Coomassie and silver staining methods, as well as fluorescent stains that are specific for phosphoproteins and glycoproteins. Staining of proteins in SDS‐polyacrylamide gels is described; variations for fluorescent staining of proteins in nondenaturing gels are also included. Support protocols describe photography of stained proteins. Curr. Protoc. Mol. Biol. 85:10.6.1‐10.6.27. © 2009 by John Wiley & Sons, Inc.

Keywords: silver; Coomassie; SYPRO; electrophoresis; protein; staining

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • 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
  • Alternate Protocol 4: Mass Spectrometry–Compatible Rapid Silver Staining
  • Support Protocol 1: Photography of Coomassie‐ or Silver‐Stained Gels
  • Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red
  • Alternate Protocol 5: Fluorescent Staining on Nondenaturing Gels Using SYPRO Orange or Red
  • Basic Protocol 4: Fluorescent Staining Using SYPRO Ruby for 2‐D Gels
  • Support Protocol 2: Photography of Fluorescently Stained Gels
  • Alternate Protocol 6: Fluorescent Staining for Selective Detection of Phosphoproteins
  • Support Protocol 3: Imaging and Documenting the Phosphoprotein‐Stained Gel
  • Alternate Protocol 7: Selective Detection of Phosphotyrosine Residues
  • Alternate Protocol 8: Fluorescent Staining for Differential Detection of Glycosylated and Nonglycosylated Proteins
  • Support Protocol 4: Viewing and Photographing the Glycoprotein‐Stained Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Coomassie Blue Staining

  Materials
  • Polyacrylamide gel (unit 10.2)
  • 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
  • Orbital shaker or rocking platform
  • 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
  • Plastic or glass container

Basic Protocol 2: Silver Staining

  Materials
  • Polyacrylamide gel (unit 10.2)
  • 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)
  • Silver nitrate solution (see recipe)
  • Developing solution (see recipe)
  • Kodak Rapid Fix Solution A
  • Plastic container
  • Orbital shaker
  • Whatman 3MM filter paper (optional)
  • Gel dryer (optional)
  • Additional reagents and equipment for photographing the gel (see protocol 7)
NOTE: Wear gloves at all times to avoid fingerprint contamination.

Alternate Protocol 2: Nonammoniacal Silver Staining

  • 5 µg/ml dithiothreitol (DTT; appendix 22)
  • 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

Alternate Protocol 4: Mass Spectrometry–Compatible Rapid Silver Staining

  Materials
  • Ethanol
  • Acetic acid
  • Ultrapure water
  • SilverQuest Silver Staining kit (Invitrogen) containing:
    • Sensitizer
    • Stainer
    • Developer
    • Developer enhancer
    • Stopper
    • Destainer A and B
  • Gel containing protein of interest
  • Microwave‐safe plastic container, preferably polypropylene
  • Microwave
  • Rotary shaker
  • Digital scanner or CCD camera

Support Protocol 1: 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
  • Small plastic dishes
  • Aluminum foil
  • Additional reagents and equipment for one‐dimensional SDS‐PAGE (unit 10.2) and photography of fluorescently stained gels (see protocol 11)

Basic Protocol 3: Fluorescent Staining Using SYPRO Orange or Red

  Materials
  • 1‐D or 2‐D polyacrylamide or IEF gel (units 10.2 10.4)
  • Fixing solution for SYPRO Ruby staining (see recipe)
  • SYPRO Ruby protein gel stain (Invitrogen; 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 11)

Alternate Protocol 5: 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 10.2 10.4)
  • Phosphoprotein standards (PeppermintStick Phosphoprotein Molecular Weight Standards, Invitrogen; also see Table 10.6.1)
  • Fixing solution for phosphoprotein gels (see recipe)
  • Pro‐Q Diamond phosphoprotein gel stain (Invitrogen; see recipe)
  • Phosphoprotein gel destaining solution (Invitrogen; see recipe)
  • 1.5‐microcentrifuge tubes
  • Vortex
  • Microcentrifuge
  • Speedvac evaporator
  • Polystyrene staining dish (e.g., a weighing dish)
  • Orbital shaker
    Table 0.6.1   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

  • Additional reagents and equipment for gel electrophoresis (units 10.2 10.4), SYPRO Ruby staining (see 10.6), Coomassie blue staining (see 10.6), silver staining (see 10.6), and imaging and documenting phosphoprotein‐stained gels (see 10.6)

Basic Protocol 4: Fluorescent Staining Using SYPRO Ruby for 2‐D Gels

  • Gel for phosphoprotein fluorescent staining (either before fixation/staining or after fixation/staining/destaining; see protocol 12)
  • 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 10.2)
  • Sample buffer (unit 10.2)
  • Pro‐Q Emerald glycoprotein gel staining kit (Invitrogen) 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 10.2), viewing and documenting glycoprotein‐stained gels (see protocol 16), and SYPRO Ruby staining (see protocol 10)
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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
   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.
   Westermeir, 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library