Different TBST pH's for WB incubation, why? - (May/18/2010 )

I've seen some protocols that use a TBST 7.5 pH for some washes and then switch over to TBS 9.5 pH, then switch back before ECL detection. Why is that?

I also saw some saying tween can react with the ECL causing background, so they wash right before with TBS (no Tween).

I use PBST (not much difference I guess with TBST), pH 7.5ish I guess. It's not too hard to try the PBS (no tween) last wash.

But I'm interested in the pH thing. The blocking and primary was carried out in pH 7.5, but the secondary was 9.5 (switching over during the washes), and then switched back before the detection (this particular one didn't use the tweenless idea).

Anybody have explanations as to why? Or is it more Lab Hocus Pocus?

It may be a stringency wash, the antibody binds better at close to pH 7, and will get partially denatured at pH 9 and so will bind less well, meaning that the stuff left on the membrane is likely to be more specific.