Electrophoretically purified KcsA heteromers. The five WT/E71A heteromers were purified by SDS-PAGE as described in Materials and methods and rerun in a 12.5% SDS polyacrylamide gel supplemented with 10 mM KCl, with or without treatment with TEV protease. The WT subunits had the long extension and the E71A subunits the short extension. Without treatment with the protease, the relative mobilities of the heteromers take on a staircase appearance because of the different total masses of the extensions. After proteolysis with TEV, all of the heteromers have the same mobility as a KcsA tetramer with an eight-Asn extension at the N terminus (right lane, M8N-KcsA; M, Met). The proteins were labeled with [³⁵S]methionine and visualized by exposure of the dried gel to x-ray film. A schematic representation of the distribution of subunits in the heterotetramers is shown (right). The table at the bottom of the figure presents the ratios of WT KcsA and E71A KcsA subunits from spontaneously dissociated tetramers (not treated with TEV protease) on the same gel. The ratios were calculated with Quantity One software (Bio-Rad Laboratories).