Native PAGE stacker pH

Dr Engelbert Buxbaum engelbert_buxbaum at
Thu Aug 24 02:55:10 EST 2006

ChenHA wrote:

> I think for some reasons, most of the people I talked to worked on
> basic proteins, perhaps that explain why they have difficulties.  So
> the question will then be, is native PAGE basically not useful for
> basic proteins?

You just have to change the buffer pH accordingly, this may also require
a change in electrical polarity. Precisely for that purpose
electrophoretic power supplies have a switch that does that.

> It is probably strange that since I work with proteins, I haven't
> actually done any native PAGE before.  But I am interested to know how
> reliable that technique is for basic proteins.  One of the problem is
> that in papers people don't report things that don't worked - for
> example, some people ran a native PAGE that gives the result that
> contradicts all the other biophysical analyses (smaller monomer rather
> than the larger oligomer expected, or vice versa).  

In native gels protein velocity depends on both charge and size, since
you have no ionic detergent that ensures equal charge/mass ratio for all
proteins. So you can not use natives for MW determination.

> The results don't
> get reported of course, but given that I have found more people who
> can't get the right result (it appears to be random process whether
> the protein bands appear in the right place), can I trust the result
> of those who use native PAGE to indicate oligomeric states for basic
> proteins?

Oligomerisation will affect size, but has little effect on charge (ionic
bond formation involves only few of the charged amino acids). Thus if
you compare the Rf values of, say, a monomer and a dimer you should get
results proportional to relative MW. However, for the reasons stated
above you can not use this method to determine absolute MWs.

I have run native gels to separate protein complexes (Hsc70/clathrin)
and then used a second dimension SDS-Gel to analyse them, this works
very well. 

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