Separation of thylakoid membrane proteins by sucrose gradient ultracentrifugation or blue native-SDS-PAGE two-dimensional electrophoresis.

Abstract

Generally, a combination of two or more chromatographic and/or electrophoretic methods is conducted to separate membrane protein complexes. Here we describe how thylakoid membrane protein complexes from the photosynthetic apparatus can be successfully separated by two main steps: preparative methods that enable purification of membrane protein complexes in the native (intact) form, and analytical methods that allow resolution of each membrane protein. Thus, separation of intact supercomplexes was achieved by solubilisation of the sample using mild detergents followed either by sucrose gradient ultracentrifugation or by blue native gel (BNG) electrophoresis. Complexes, thus, recovered were then resolved further using either reversed phase liquid chromatography or SDS-PAGE respectively.