Abstract
The ability of two-dimensional electrophoresis (2-DE), based on the original method developed more than 20 years ago (1), to separate simultaneously up to several thousand proteins using large-format gels (2) has made it the method of choice for the analysis of protein expression in complex biological systems, such as whole cells, tissues, and organisms. Initially, 2-DE only yielded information on the charge (pI), size (M r), and relative abundance of the separated proteins. However, in recent years, a variety of methods have been developed that make it possible to identify and characterize proteins separated by 2-DE. Many of these methods depend on the technique of Western electroblotting in which proteins separated by 2-DE are transferred (“blotted”) by the application of an electric field perpendicular to the plane of the gel onto the surface of an inert membrane, such as nitrocellulose (3). Methods for electroblotting of protein from 2-DE gels are described in Chapter 35.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Klose, J. and Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034–1059.
Towbin, H., Staehelin, T., and Gordon, G. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Pennington, S. R., Wilkins, M. R., Hochstrasser, D. F., and Dunn, M. J. (1997) Proteome analysis: From protein characterisation to biological function. Trends Cell. Biol. 7, 168–173.
Dunn, M. J. (1997) Quantitative two-dimensional gel electrophoresis: From proteins to proteomes. Biochem. Soc. Trans. 25, 248–254.
Hancock, K. and Tsang, V. C. W. (1983) India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133, 157–162.
Moeremans, M., Daneels, G., and De Mey, J. (1985) Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. 145, 315–321.
Patton, W., Lam, L., Su, Q., Lui, M., Erdjument-Bromage, H., and Tempst, P. (1994) Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting. Anal. Biochem. 220, 324–335.
Shojaee, N., Patton, W. F., Lim, M. J., and Shepro, D. (1996) Pyrogallol red-molybdate; A reversible, metal chelate stain for detection of proteins immobilized on membrane supports. Electrophoresis 17, 687–693.
Patterson, S. D., Hess, D., Yungwirth, T., and Aebersold, R. (1992) High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane. Anal. Biochem. 202, 193–203.
Moeremans, M., De Raeymaeker, M., Daneels, G., and De Mey, J. (1986) FerriDye: colloidal iron binding followed by Pearl’s reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes. Anal. Biochem. 153, 18–22.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Dunn, M.J. (1999). Detection of Total Proteins on Western Blots of 2-D Polyacrylamide Gels. In: Link, A.J. (eds) 2-D Proteome Analysis Protocols. Methods in Molecular Biology, vol 112. Humana Press. https://doi.org/10.1385/1-59259-584-7:319
Download citation
DOI: https://doi.org/10.1385/1-59259-584-7:319
Publisher Name: Humana Press
Print ISBN: 978-0-89603-524-9
Online ISBN: 978-1-59259-584-6
eBook Packages: Springer Protocols