anticipated, KM2 cells did not express b subunit. Western blot analysis confirmed that b

subunits without a histidine epitope tag were washed off the resin during the wash steps

(Figure 2-4B, Lanes 2 and 3) whereas b+7-his WaS retained by the Ni-resin (Figure 2-4B,

Lane 4). In lane 5, when individual membrane preparations of KM2 individually

expressing either the short or long subunit were mixed together, as expected, only the

histidine epitope tagged b subunit was purified from the Ni-resin. In the KM2 cells

expressing both b+7-his and bA7-HA, Only the histidine-tagged long b subunit appeared,

suggesting the different b subunits were segregated into separate enzyme complexes.

However, there were two important caveats: the enzyme complex may have dissociated

before coming off the Ni-resin, and a difference of fourteen amino acids may have simply

been too much for the stability of the enzyme. Both issues were studied. First, two

additional detergents to solublize the membrane were investigated. The capability of the

detergents to allow the enzyme to come off the resin intact was investigated by cross-

linking the b subunits after solubilization and Ni-resin purification and then visualized by

Western blots. Secondly, the size difference of the two b subunits was decreased by

testing for the ability of the b+7-his subunit to dimerize with a bwt-HA Subunit.

 Membrane preparations of KM2 (Ab) expressing b+7-hi WeTO Solubilized with 0.2%

solutions of tegamineoxide WS-35 (AO), taurodeoxycholate (TD), lauryldimethylamine

oxide (LDAO) or SDS and allowed to incubate at room temperature for 30 minutes. AO,

TD and LDAO were detergents commonly used to solubilize membrane proteins. LDAO

was likely to dissociate Fl from Fo. In theory, however, Fo should have remained intact

through the purification. The solubilized proteins were then mock treated (-) or treated

(+) with the homobifuctional crosslinker, BS3. The cross-linking reaction was stopped by