Membtrane prep. Ni-Resin Purified





 BS3- -- + + + +
 A -- b


 anti-b b

 1 234 56$7 8 9 1011 12
 B :


 a nti-V5 r


Figure 2-1 1. Ni-resin purification of F,Fo ATP synthase treated with the cross-linker BS3
 Crude membrane preparations of KM2 expressing the normal length b
 subunits with epitope tags were mock treated (-) or treated (+) with 1mM
 bis(3 -sulfo-N -hydroxysuccinimide ester) (B S3) for 20 minutes at room
 temperature. The cross-linking reaction was stopped by addition of 100 mM
 ethanolamine HC1, pH 7.5 for 10 minutes. The products were then solubilized
 with 0.2% tegamineoxide WS-3 5 and subj ected to Ni-resin purification before
 separation on a 15% polyacrylamide Tris-glycine SDS gel and subsequent
 Western blot analysis. A) Proteins were transferred to a nitrocellulose
 membrane and probed using a polyclonal anti-b antibody. B) Proteins were
 transferred to a nitrocellulose membrane and probed using an anti-V5 mouse
 monoclonal antibody.