Summary
Numerous protein detection and quantitation methods for gel-based proteomics have been devised that can be classified in three major categories: (1) Universal (or “general”) detection techniques, which include staining with anionic dyes (e.g., Coomassie brilliant blue), reverse (or “negative”) staining with metal cations (e.g., imidazole-zinc), silver staining, fluorescent staining or labeling, and radiolabeling, (2) specific staining methods for the detection of post-translational modifications (e.g., glycosylation or phosphorylation), and (3) differential display techniques for the separation of multiple, covalently tagged samples in a single two-dimensional electrophoresis (2-DE) gel, followed by consecutive and independent visualization of these proteins to minimize methodical variations in spot positions and in protein abundance, to simplify image analysis, as well as to improve protein quantitation by including an internal standard.The most important properties of protein detection methods applied in proteome analysis include high sensitivity (i.e., low detection limit), wide linear dynamic range for quantitative accuracy, reproducibility, cost-efficiency, ease of use, and compatibility with downstream protein identification or characterization technologies, such as mass spectrometry (MS). Regrettably, no single detection method meets all these requirements, albeit fluorescence-based technologies are currently favored for most applications; hence, the major focus of this chapter is on fluorescent-dye-based protein detection and quantitation techniques. Although satisfying results with respect to sensitivity and reproducibility are also obtained by methods based on radioactive labeling of proteins (which is still unsurpassed in terms of sensitivity), radiolabeling is, however, largely impractical for routine proteomic profiling because of the costs and the health and safety concerns associated with handling radioactive compounds.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Görg, A., Weiss, W., and Dunn, M.J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685. Review.
Patton, W.F. (2002) Detection technologies in proteome analysis. J. Chromatogr. B 771, 3–31. Review.
Van den Bergh, G., and Arckens, L. (2004) Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Curr. Opin. Biotech. 15, 38–43. Review.
Miller, I., Crawford, J., and Gianazza, E. (2006) Protein stains for proteomic applications: which, when, why? Proteomics 6, 5385–5408. Review.
Harris, L.R., Churchward, M.A., Butt, R.H., and Coorssen, J.R. (2007) Assessing detection methods for gel-based proteomic analyses. J. Proteome Res. 6, 1418–1425.
Fazekas de St.Groth, S., Webster, R.G., and Datyner, A. (1963) Two new staining procedures for quantitative estimation of proteins on electrophoresis strips. Biochim. Biophys. Acta 71, 377–391.
Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.
Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, 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.
Luo, S., Wehr, N.B., and Levine, R.L. (2006) Quantitation of protein on gels and blots by infrared fluorescence of Coomassie Blue and Fast Green. Anal. Biochem. 350, 233–238.
Fernandez-Patron, C., Castellanos-Serra, L., and Rodriguez, P. (1992) Reverse staining of sodium dodecyl sulfate polyacrylamide gels by imidazole-zinc salts: sensitive detection of unmodified proteins. Biotechniques 12, 564–573.
Castellanos-Serra, L., and Hardy, E. (2006) Negative detection of biomolecules separated in polyacrylamide electrophoresis gels. Nat. Protoc. 1, 1544–1551.
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.
Merril, C.R., Goldman, D., Sedman, S.A., and Ebert, M.H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437–1438.
Blum, H., Beier, H., and Gross, H.J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99.
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.
Chevallet, M., Luche, S., and Rabilloud, T. (2006) Silver staining of proteins in polyacrylamide gels. Nat. Protoc. 1, 1852–1858.
De Moreno, M.R., Smith, J.F., and Smith, R.V. (1985) Silver staining of proteins in polyacrylamide gels: increased sensitivity through a combined Coomassie Blue-silver stain procedure. Anal. Biochem. 151, 466–470.
Becher, B., Knofel, A.K., and Peters, J. (2006) Time-based analysis of silver-stained proteins in acrylamide gels. Electrophoresis 27, 1867–1873.
Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858.
Mortz, E., Krogh, T.N., Vorum, H., and Görg, A. (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1, 1359–1363.
Harris, L.R., Churchward, M.A., Butt, R.H., and Coorssen, J.R. (2007) Assessing detection methods for gel-based proteomic analyses. J. Proteome Res. 6, 1418–1425.
Ünlü, M., Morgan, M.E., and Minden, J.S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077.
Berggren, K.N., Schulenberg, B., Lopez, M.F., Steinberg, T.H., Bogdanova, A., Smejkal, G., Wang, A., and Patton, W.F. (2002) An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation. Proteomics 2, 486–498.
Mackintosh, J.A., Choi, H.Y., Bae, S.H., Veal, D.A., Bell, P.J., Ferrari, B.C., Van Dyk, D.D., Verrills, N.M., Paik, Y.K., and Karuso, P. (2003) A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis. Proteomics 3, 2273–2288.
Rabilloud, T., Strub, J.M., Luche, S., van Dorsselaer, A., and Lunardi, J. (2001) A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. Proteomics 1, 699–704.
Lamanda, A., Zahn, A., Röder, D., and Langen, H. (2004) Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution. Proteomics 4, 599–608.
Reinders, J., and Sickmann, A. (2005) State-of-the-art in phosphoproteomics. Proteomics 5, 4052–4061. Review.
Steinberg, T.H., Agnew, B.J., Gee, K.R., Leung, W.Y., Goodman, T., Schulenberg, B., Hendrickson, J., Beechem, J.M., Haugland, R.P., and Patton, W.F. (2003) Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3, 1128–1244.
Hart, C., Schulenberg, B., Steinberg, T.H., Leung, W.Y., and Patton, W.F. (2003) Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24, 588–598.
Wu, J., Lenchik, N.J., Pabst, M.J., Solomon, S.S., Shull, J., and Gerling, I.C. (2005) Functional characterization of two-dimensional gel-separated proteins using sequential staining. Electrophoresis 26, 225–237.
Alban, A., David, S.O., Bjorkesten, L., Andersson, C., Sloge, E., Lewis, S., and Currie, I. (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44.
Shaw, J., Rowlinson, R., Nickson, J., Stone, T., Sweet, A., Williams, K., and Tonge, R. (2003) Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3, 1181–1195.
Lilley, K.S., and Friedman, D.B. (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Rev. Proteomics 1, 401–409.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Weiss, W., Weiland, F., Görg, A. (2009). Protein Detection and Quantitation Technologies for Gel-Based Proteome Analysis. In: Reinders, J., Sickmann, A. (eds) Proteomics. Methods in Molecular Biology™, vol 564. Humana Press. https://doi.org/10.1007/978-1-60761-157-8_4
Download citation
DOI: https://doi.org/10.1007/978-1-60761-157-8_4
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-156-1
Online ISBN: 978-1-60761-157-8
eBook Packages: Springer Protocols