Analysis of Thylakoid Protein Complexes by Two-Dimensional Electrophoretic Systems

Photosynthetic machinery in the thylakoid membrane is prone to modifications depending on �environmental, developmental, and morphological parameters. Such plasticity in the composition of the thylakoid membrane protein complexes guarantees efficient function of the photosynthetic machinery. In this chapter, we describe methods for separation of thylakoid membrane protein complexes at high resolution by two-dimensional gel electrophoretic systems. Solubilization of the thylakoid membrane protein complexes either by dodecylmaltoside or digitonin is described first. Then, two partially overlapping �methods, blue native gel electrophoresis and high-resolution clear native gel electrophoresis, are demonstrated to separate the individual protein complexes. Finally, denaturing SDS-polyacrylamide gel electrophoresis is used to reveal the protein composition of each complex. Critical points in all protocols are addressed and representative examples of the composition of Arabidopsis thaliana thylakoid membrane protein complexes are shown.