92 A Membrane b+~7-his Preparation bb7H B Ni-Purifiedb+-i Protein Figure 2-4. Western blot analysis of histidine and HA-epitope tagged b subunits. A) KM2 (Ab) cells were transformed with pKAM14 (be), pTAM34 (bA7-HA), pTAM3 5 (b+7-his), or both pTAM34 and pTAM3 5. Crude membrane preparations were made and proteins separated on a 15% polyacrylamide Tris- HCl-SDS BioRad Ready gel. The proteins were transferred to a nitrocellulose membrane and probed with anti-b anitbodies. Lane 5 represents a mixture of membranes in lanes 3 and 4. B) The crude membrane preparations were solubilized with 0.2% LDAO and then subj ected to Ni-resin purification under native conditions (see Materials and Methods). The purified proteins were then separated with SDS-PAGE and Western blotted with anti-b antibodies. Ni-Resin Purification Initial experiments utilizing the two plasmid expression system approach indicated that the two different b subunits were incorporated into F1Fo ATP synthase in a segregated manner, suggesting that the b subunit dimer must form in parallel during F1Fo ATP synthase assembly. The two b subunits, expressed from pTAM35 (b+7-his) and pTAM34 (bA7-HA), were expressed in the same KM2 (Ab) cell and their ability to exist in the same F1Fo ATP synthase enzyme complex was determined by Ni-resin purification followed by Western blot analysis (Figure 2-4B). Upon Ni-resin purification, only enzyme containing the histidine epitope tagged b subunit should have been present. As