The working principle of BN/SDS-PAGE. A) After solubilization, the mixture of different protein complexes is separated by BN-PAGE, according to their molecular weight. B) Following the gel run, the lane of the gel is excised and subjected to a denaturation solution (eg 1% SDS and 1% β-mercaptoethanol) so that the native protein complexes (underlayed in grey colour) dissociate to their constituent polypeptides. Loosely stuck in the pores of the gel, the subunits of the protein complexes (underlayed in red colour) remain at their position until they are forced electrophoretically into the second dimension gel. Due to the SDS used in the denaturation step (and residual Coomassie) the polypeptides are uniformly negatively charged and are separated according to their molecular weight in the gel. Subunits of a protein complex form a vertical row on the second dimension gel.