The hierarchical
composition and interactions of the labile thylakoid protein complexes can be
assessed by sequential 2D-native gel-electrophoresis system. Mild non-ionic
detergent digitonin is used to solubilize labile protein super-and
megacomplexes, which are then separated with first-dimension blue native
polyacrylamide gel electrophoresis (1D-BN-PAGE). The digitonin derived protein
complexes are further solubilized with stronger detergent, β-DM, and subsequently separated on an orthogonal
2D-BN-PAGE to release smaller protein subcomplexes from the higher-order
supercomplexes. Here we describe a detailed method for 2D-BN-PAGE analysis of
thylakoid protein complexes from Arabidopsis thaliana.