M~mt~ran~ pr~p. Ni-


 -a a
 o

 a~L
 ~I vlvl
ru 5. C r > > c r




 ~rr~t:


Resin Purified


A

 anti-b


% membrane %
 b-his


membrane
 b-VS


wt/wt


51 &4


49 & 4

54+ 4

56 + 6


15 A1

11 &2

4 11


+7/wt 46 + 4

+7/A7 44 + 6


Figure 2-13. Quantitation of b subunit heterodimeric F1Fo. Membranes were prepared
 from IPTG-induced cells by differential centrifugation. A total of 15 mg
 membrane protein was solubilized, Ni-resin purified (1 ml packed resin
 volume), concentrated with a Microcon YM-10 and separated on a 15 cm 15%
 polyacrylamide Tris SDS gel to allow maximal separation. A) Proteins were
 transferred to a nitrocellulose membrane and probed using a polyclonal anti-b
 antibody. This Western blot was deliberately overexposed to allow easy
 visualization of the V5-tagged b subunits in the Ni-resin purification lanes. B)
 Densitometric analysis of lower exposures was used to determine the relative
 amounts of histidine-tagged and V5-tagged b subunits in samples
 coexpressing the different subunits. Reletive amounts of b subunits were
 determined for crude membrane preparations and Ni-resin purified samples
 coexpressing both b subunits. The percent of heterodimer formation was
 calculated based on three independent membrane preparations as well as three
 separate exposure times.


% Ni-Purified
heterodimer