Native PAGE stacker pH

ChenHA hzhen at freeuk.com
Sat Aug 19 03:25:22 EST 2006



DK wrote:
> 
> Just a minor terminology confusion. "Drop" above is a verb, not noun.
> Voltage drops *on* stacker (The analogy is river with a waterfall).
> Most of the "drop" occurs where resistance is highest, e.g. stacker.
> So it is the electric field *intensity* (which is a driving force of
> ectrophoretic movement), that is high in the stacker. If under "local
> voltage" you meant difference in electric potentials between stacker
> ends, then you are absolutely correct - it will be larger than the
> difference of potentials between, say, ends of separating gel or
> power supply end the start of stacker.

OK, thanks for the clarification.  I thought you meant the voltage is
lower when you said "drops". 


> IMHO, it's pretty straightforward but heavily depends on your protein.
> In many cases, a particular protein is unstable/aggregating
> in the buffer conditions chosen (true particularly for many loading
> buffers). For most acidic proteins, it's really a piece of cake - 

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?

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).  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?


> just use
> you regular SDS-PAGE system and omit SDS from everywhere.
> It goes without saying that the gel has to be run in the cold room, with
> ice cold buffers and either be cooled with the recirculating bath (as Hoefer
> allows) or be completely submersed in large volume of the lower buffer
> (as possible with Bio-Rad's mini). Also run at lower voltage (I use 150V
> for Bio-Rad) and runs take much longer, particularly if you want
> separation between specific proteins.
> 
> No matter what, native gels always look a lot uglier than SDS-PAGE.
> I find natives indispensable in refolding studies - you can clearly see the
> amout of oligomers, smearing, etc and estimate refolding efficiency.
> Being able to do it for 26 sample at a time surely beats running
> 26 HPLC gel-filtrations... (There are some pretty neat things that
> can be observed. For example, in one case I clearly that glycerol
> and Triton X-100 stabilized some intermediate fold that ran right
> in between a properly folded monomer and some weird dimer).
> 
> DK


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