Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

Abstract

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-β- d-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-β- d-maltoside and separated in 4–7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-β- d-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5–20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3′,5,5′-tetramethylbenzidine-H 2O 2, Coomassie blue, and silver staining. A number of protein components were identified on “Western blots” of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a b protein complex, photosystem I, photosystem II, the cytochrome b 6 f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.