Protein Detection in Gels Without Fixation

Won‐A Joo1, David W. Speicher1

1 The Wistar Institute, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.6
DOI:  10.1002/0471140864.ps1006s48
Online Posting Date:  May, 2007
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Abstract

A number of alternative methods are described for detecting proteins in polyacrylamide gels that do not require fixation of the protein either prior to staining or in conjunction with staining. The primary advantage of avoiding fixation is that this makes it easier to remove proteins of interest from the gels for subsequent analysis. In general, the sensitivity of protein detection methods that avoid fixation is lower than for detection methods using fixation. For any given method, sensitivity is dependent on the volume of the protein band within the gel; hence, sensitivity is highest for sharp, narrow bands. Techniques described in this unit include protocols for protein detection in gels by SDS precipitation, preparation of contact blots, staining with imidazole‐zinc, and use of the fluorescent labels IAEDANS and fluorescamine. Several additional methods, including the use of tryptophan fluorescence, guide strips, and minimal protein staining, are discussed in the Commentary.

Keywords: protein detection; polyacrylamide gels; protein stains; SDS precipitation; contact blots; imidazole‐zinc staining; fluorescent labeling

     
 
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Table of Contents

  • Basic Protocol 1: Protein Detection in Gels by SDS Precipitation
  • Basic Protocol 2: Preparation of Contact Blots
  • Basic Protocol 3: Imidazole‐Zinc Staining of Proteins
  • Basic Protocol 4: Iaedans Modification of Proteins
  • Basic Protocol 5: Fluorescamine Labeling of Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Protein Detection in Gels by SDS Precipitation

  Materials
  • SDS‐polyacrylamide gel containing protein of interest (units 10.1to 10.4)
  • 4 M sodium acetate, pH unadjusted ( appendix 2E; store at 4°C; stable up to 6 months)
  • Black, nonreflective surface
  • Fluorescent desk lamp
NOTE: All steps are performed at room temperature.

Basic Protocol 2: Preparation of Contact Blots

  Materials
  • Polyacrylamide gel containing protein of interest (units 10.1to 10.4)
  • 100% methanol
  • 1× transfer buffer (see recipe)
  • PVDF transfer membrane: 0.2‐µm polyvinylidene difluoride (PVDF) membrane (Bio‐Rad)
  • Whatman no. 1 filter paper
  • Additional reagents and equipment for staining membranes (unit 10.8)
NOTE: Use powder‐free gloves rinsed in Milli‐Q water when handling all materials for this procedure, and handle membranes with forceps at the edges to avoid potential artifactual staining.

Basic Protocol 3: Imidazole‐Zinc Staining of Proteins

  Materials
  • Polyacrylamide gel containing protein of interest (units 10.1to 10.4)
  • 0.2 M imidazole solution containing 0.1% (w/v) SDS, pH is not adjusted
  • 0.2 M zinc sulfate solution
  • Transparent plastic tray

Basic Protocol 4: Iaedans Modification of Proteins

  Materials
  • Protein sample (<5 mg protein in 0.3‐ to 3‐ml volume)
  • 0.75 M Tris⋅Cl (pH 8.6)/15 mM EDTA (make with 4°C water, measure pH at that temperature, and use within 1 week; store at 4°C)
  • Ultrapure Urea (Bio‐Rad)
  • Dithiothreitol (DTT or Cleland's reagent; Calbiochem)
  • Argon or nitrogen gas
  • N‐Iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethylenediamine (IAEDANS; Sigma; keep light yellow crystals desiccated in the dark at −20°C)
  • 2‐mercapthoethanol
  • Additional reagents and equipment for dialysis ( appendix 3B) and electrophoresis (units 10.1to 10.4)
CAUTION: Use UV‐rated eye protection when using UV lamps and minimize skin exposure to direct or reflected UV light.

Basic Protocol 5: Fluorescamine Labeling of Proteins

  Materials
  • Protein sample (0.5 to 1 mg/ml)
  • 15 mM sodium phosphate buffer, pH 8.5 ( appendix 2E; prepare with 4°C water just before use)
  • SDS (Bio‐Rad)
  • Sucrose
  • Dithiothreitol (DTT; optional)
  • 1 mg/ml fluorescamine (Sigma) in acetone
  • 5 mg/ml bromphenol blue (optional)
  • 37°C water bath
  • Additional reagents and equipment for dialysis ( appendix 3B) and electrophoresis (unit 10.1)
CAUTION: Use UV‐rated eye protection when using UV lamps and minimize skin exposure to direct or reflected UV light.
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Figures

Videos

Literature Cited

   Eng, P.R. and Parkes, C.O. 1974. SDS electrophoresis of fluorescamine‐labeled proteins. Anal. Biochem. 59:323‐325.
   Hardy, E. and Castellanos‐Serra, L.R. 1992. Reverse staining of sodium dodecyl sulfate‐polyacrylamide gels by imidazole‐zinc salts: Sensitive detection of unmodified proteins. BioTech. 12:564‐573.
   Higgins, R.C. and Dahmus, M.E. 1979. Rapid visualization of protein bands in preparative SDS‐polyacrylamide gels. Anal. Biochem. 93:257‐260.
   Speicher, D.W., Davis, G., and Marchesi, V.T. 1983. Structure of human erythrocyte spectrin. II. The sequence of the α‐I domain. J. Biol. Chem. 258:14938‐14947.
   Towbin, H., Staehlin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76:4350‐4354.
   Wallace, R.W., Yu, P.H., Dieckert, J.P., and Dieckert, J.W. 1974. Visualization of protein‐SDS complexes in polyacrylamide gels by chilling. Anal. Biochem. 61:86‐92.
Key Reference
   Hames, B.D. and Rickwood, D. (eds.) 1990. Gel Electrophoresis of Proteins: A Practical Approach. Oxford University Press, New York.
  A general reference for gel electrophoresis and protein detection methods.
   Simpson, R. 2003. Peptide mapping and sequence analysis of gel‐resolved proteins. In Protein and Proteomics: A Laboratory Manual (R. Simpson, ed.) pp. 394‐395. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  A general reference for gel electrophoresis and protein detection methods.
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