Separation of Plant Proteins by Electrophoresis

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 ).