Summary
Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. “Detection” precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.
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References
Salinovich, O. and Montelaro, R.C. (1986) Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem. 156, 341–347.
Klarskov, K. and Naylor, S. (2002) India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom. 16, 35–42.
Yonan, C.R., Duong, P.T., and Chang, F.N. (2005) High-efficiency staining of proteins on different blot membranes. Anal Biochem. 338, 159–161.
Moeremans, M., Daneels, G., and De Mey, J. (1985) Sensitive colloidal metal (gold or silver) staining of proteins blots on nitrocellulose membranes. Anal Biochem. 145, 315–321.
Fazekas de St Groth, S., Webster, R.G., and Datyner, A. (1963) Two new staining procedures for quantitative estimation of proteins in electrophoresis strips. Biochim Biophys Acta. 71, 377–391.
Anderson, N.L., Esquer-Blasco, R., Hofmann, J.-P., and Anderson, N.G. (1991) A two-dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies. Electrophoresis 12, 907–930.
Gorovsky, M.A., Carlson, K., and Rosenbaum, J.L. (1970) Simple method for quantitive densitometry of polyacrylamide gels using fast green. Anal Biochem. 35, 359–370.
Allen, R.E., Masak, K.C., and McAllister, P.K. (1980) Staining protein in isoelectric focusing gels with fast green. Anal Biochem. 104, 494–498.
Syrovy, I. and Hodny, Z. (1991) Staining and quantification of proteins separated by polyacrylamide gel electrophoresis. J Chromatogr. 569, 175–196.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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D’souza, A., Scofield, R.H. (2009). Protein Stains to Detect Antigen on Membranes. In: Kurien, B., Scofield, R. (eds) Protein Blotting and Detection. Methods in Molecular Biology, vol 536. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-542-8_43
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DOI: https://doi.org/10.1007/978-1-59745-542-8_43
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