Native PAGE stacker pH
Dr Engelbert Buxbaum
engelbert_buxbaum at hotmail.com
Thu Aug 24 02:55:10 EST 2006
> 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
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
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