40] Electrophoretic analysis of chloroplast proteins

Abstract

Publisher Summary This chapter discusses the electrophoretic analysis of chloroplast proteins. Electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gels has become a standard technique for structural and biosynthetic studies of chloroplast proteins. A number of electrophoretic systems with different degrees of resolution have been employed in such investigations. This chapter describes a method which combines the alkaline SDS-discontinuous buffer system of Neville and, in the resolving gel, a linear acrylamide concentration gradient. The discontinuous buffer system is capable of stacking SDS–protein complexes over a wide range of molecular weights, thus providing very sharp bands, while the linear pore gradient allows the separation and resolution of polypeptides with widely different molecular weights. The combination of these two methods gives consistently high resolution of thylakoid membrane polypeptides and chloroplast stromal proteins. For optimal resolution, approximately 20 cm of resolving gel and 1.5–2.0 cm of stacking gel should be used. The acrylamide concentration of the stacking gel may be decreased to 4 or 5% depending on the molecular weight distribution of the polypeptide mixture to be analyzed.