Native PAGE stacker pH

[Dr Engelbert Buxbaum]

DK wrote:


> This made me thinking: In my experience, CHAPS and 
> cholate/desoxycholate are mild enough to keep most 
> non-membrane proteins and many of their complexes intact. Still,
> they are charged detergents and their inclusion into native 
> gels may improve resolution and decrease smearing. 

CHAPS has both a negative and a positive charge, so it will not
influence electrophoretic behaviour. Indeed, that's why it is used in
IEF. Use of cholate and its derivatives in PAGE instead of SDS should be
possible, I have never heard of anybody doing it.

> Also, Engelbert, can you recommend a reliable buffer system
> for basic proteins? Personally, I don't believe simply reversing 
> polarity on Ornstein and Davis is going to produce satisfactory 
> results in most cases.

No, you need to change the buffer pH and buffering ions as well, to get
a proper stacking effect, as described by Jovin. Apart from the
literature I cited in earlier posts you may check my own paper in Anal.
Biochem. 314 (2003) 70-76 for recipes for cationic electrophresis. This
works well not only for glycoproteins (for which I developed the method)
but also with nuclear proteins. It is certainly possible to use the
buffer system also without CTAB.