Abstract
Direct identification of the product of a transferred gene is clearly an important part of the characterization of a transgenic plant. It is possible to detect proteins using highly sensitive ELISA techniques (Chapter 36, this volume), and to determine their precise histological or subcellular locations using immunohistochemistry (Chapter 37, this volume). However, it is essential that the expressed protein should also be separated by electrophoresis, and detected either by staining or Western blot analysis. Selection of the most suitable electrophoresis system will allow the researcher to determine the M r of the protein in the native and denatured states, its isoelectric point and charge at various pH values, its interactions (via disulfide bonds) with other subunits, and the presence or absence of glycosylation. This will, in fact, confirm whether the protein has been correctly processed, or whether any proteolysis or incorrect post-translational processing has occurred. Furthermore, it is possible to confirm that the protein has the correct N-terminal amino acid sequence by transfer to a PVDF based membrane for microsequencing. The present chapter describes methods for the extraction, electrophoresis, and detection of proteins, and for their transfer to membranes for microsequencing. These can be used for the direct analysis of expressed proteins, or combined with Western blotting as described in the following chapter (Chapter 35).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 680–685.
Davis, B. J. (1964) Disc Electrophoresis—II Method and application to human serum proteins. Ann. NY Acad Sci. 121, 404–427.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of protein. J. Biol Chem. 250, 4007–4021.
Blakesley, R. W. and Boezi, J. A. (1977) A new staining technique for proteins in polyacrylamide gels using Coomassie brilliant blue G250. Anal. Biochem. 82, 580–582.
Morrissey, J. H. (1981) Silver stain for proteins m polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117, 307–310.
Nelson, T., Harpster, M. H., Mayfield, S. P., and Taylor, W C. (1984) Light regulated gene-expression during maize leaf development. J. Cell Biol. 98, 558–564.
de Jong, W. W., Zweers, A., and Conen, L. H. (1978) Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulphate-protein complexes. Biochem. Biophys. Res. Commun. 82, 532–539.
Schägger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulphate-poly-acrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal. Biochem. 166, 368–379.
Koenig, R., Stegemann, H., Francksen, H., and Paul, H. L. (1970) Protein subunits in the potato virus N group. Determination of the molecular weights by polyacrylamide electrophoresis. Biochem. Biophys. Acta. 207, 184–189.
Ornstein, L. (1964) Disc Electrophoresrs—I. Background and theory. Ann. NY Acad Sci. 121, 321–346.
Hames, B. D. (1990) In Gel electrophoresis of Proteins: A practical approach, 2nd ed. (Hames, B. D., and Rickwood, D., ed.), IRL Press, Oxford, pp. 1–147.
Clements, R L. (1987) A study of gliadins of soft wheats from the Eastern United States using a modified polyacrylamide gel electrophoresis procedure. Cereal Chem. 64, 442–448.
Righetti, P. G., Gianazza, E., Viotti, A., and Soave, C. (1977) Heterogeneity of storage proteins of maize. Planta. 136, 115–123.
O’Farrell, P. Z., Goodman, H. M., and O’Farrell, P. H. (1977) High resolution two dimensional electrophoresis of basic as well as acidic proteins. Cell. 12, 1133–1142.
Lee, C., Levin, A., and Branton, D. (1987) Copper staining. A five-minute protein stain for sodium dodecyl sulphate-polyacrylamide gels. Anal. Biochem. 166, 308–312.
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.
Swrtzer, 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.
Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) A simplified ultrasensitive silver stain for detecting proteins m polyacrylamide gels. Anal. Biochem. 105, 361–363.
Moreno, De 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.
Gottlieb, M. and Chavko, M. (1987) Silver staining of native and denatured eucaryotic DNA in agarose gels. Anal. Biochem. 165, 33–37.
Chuba, P. J. and Palchaudhuri, S. (1986) Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels. Anal. Biochem. 156, 136–139.
Sargent, J. R. and George, S. G. (1975) in Methods in Zone Electrophoresis, BDH Chemicals Ltd., Poole, UK.
Zaccharius, R. J., Zell, T. E., Morrison, J. H., and Woodlock, J. J. (1969) Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 31, 148–152.
Yuen, S. W., Chui, A. H., Wilson, K. J., and Yuan, P. M. (1988) Microanalysis of SDS-PAGE electroblotted proteins, in Applied Biosystems Protein Sequences User Bulletin No. 36.
Bunce, N. A. C., White, R. P., and Shewry, P. R. (1985) Variation in estimation of molecular weights of cereal prolamins by SDS-PAGE. J. Cereal Sci. 3, 131–142.
Baker, C. S., Dunn, M. J., and Yacoub, M. H. (1991) Evaluation of membranes used for electroblotting of proteins for direct automated microsequencing. Electrophoresis 12, 342–348.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1995 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Shewry, P.R., Tatham, A.S., Fido, R.J. (1995). Separation of Plant Proteins by Electrophoresis. In: Jones, H. (eds) Plant Gene Transfer and Expression Protocols. Methods in Molecular Biology™, vol 49. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-321-X:399
Download citation
DOI: https://doi.org/10.1385/0-89603-321-X:399
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-321-4
Online ISBN: 978-1-59259-536-5
eBook Packages: Springer Protocols