Western Analysis of Transgenic Plants

Abstract

Western blotting combines the resolving power of protein electrophoresis and the specificity of immunology in a rapid and sensitive format for the identification of proteins in complex mixtures. Proteins resolved by electrophoresis are transferred to a solid support, which is normally nitrocellulose or polyvinylidene fluoride (PVDF [Millipore, Bedford, MA]). Immunological detection of proteins transferred to a solid support combines ease of handling with accessibility of antibodies to the immobilized protein. A primary antibody is bound to a specific antigen on the membrane, and this antibody is detected using a labeled high-affinity reporter (frequently an enzyme-linked antibody). This chapter describes a basic protocol for the Western blotting and detection of transgene expression products in plants (Fig. 1). An alternative procedure for the initial immunopurification and concentration of a transgene product is also described, based on the use of affinity-purified antibodies covalently attached to magnetic beads. Western blotting can be applied to any gel separation technique; however, one-dimensional sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) (1,2) is most commonly used.