native PAGE pH

[Nicola Traverso]

Dear Dr Engelbert Buxbaum

I found this e-adress in bio.net.
I'm a little bit confused about pH in PAGE gels, in particular native 
gels. I need your help.

When in SDS-PAGE (discontinuous system) I use
-tris-glycine pH 8.3 as electrophoretic buffer
-tris-HCl pH 6.8 as stacking buffer
-tris-HCl pH 8.8 as resolving buffer
at which pH are proteins separated?
I though at 8.8, i.e. the pH of the resolving buffer, but I've recently 
read that they separate at pH 9.5! How can this be true?
Does the pH of the tris-glycine electrophoretic buffer influence the pH 
of the resolving gel during the electrophoretic run? if yes, how much?
 
In native-PAGE (continuous system)
-do I have to use the same pH in sample buffer, electrophoretic buffer 
and resolving buffer? Tris-HCl is OK? Must I use tris-glycine in the 
electrophoretic buffer? May I change the pH, let us say, between 7 and 
10 as I like?

In native-PAGE (discontinuous system)
-how can I set systems at pH different than usual (stacking: 6.8; 
resolving 8.8; electrophoretic 8.3)? for exemple, if I desire to resolve 
at pH 7.8, how do I have to prepare the other buffers (stacking, sample, 
and electrophoresis)?

Thank you in advance

Best regards

Nicola Traverso
Dept Experimental Medicine
Section of General Pathology
University of Genova
Italy