How to show protein interaction?

[Andy Phillips]

Achim Recktenwald, PhD wrote:

> > Thorsten Schmidt (Thorsten.Schmidt at rz.ruhr-uni-bochum.de) wrote:
> > > I have a protein and I just want to test if it forms dimers, trimers etc.?
> > > (The protein was purified by HisTag-Chromatographie and is in solution
> > > (20mM Tris, 0,5 M NaCl, 1 M Imidazol))
> > > What is the simplest way to do this?

> I would also try native polyacrylamide-gel-electrophoresis. If the bond
> between the subunits is strong, you'll get one single band, the multimer
> form; if its very weak, you'll only see a sharp band at the mol. weight
> for the single subunit. But if the bond is neither nor than you get a
> smear on your gel, starting at the point of the highest multimer down to
> the lowest multimer, or even the single subunit. In the latter case the
> multimer form is constantly decomposing during the electrophoresis.
> 
> Achim

Non-denaturing PAGE is not to be trusted for estimation of molecular weights: in 
electrophoresis, the rate at which proteins move is in inverse relationship to 
their mol. weight, because the bound SDS makes all proteins have roughly the same 
charge/mass ratio. In the absence of SDS, the charge on the protein is a function 
of its pI, so two proteins with the same mass but different pIs will move at 
different rates: the protein with low pI have a higher net negative charge than 
that with a high pI, and so the former will move faster in the gel. As an extreme 
example, cytochrome c (pI ~10) in a non-denaturing PAGE gel at pH8 will move 
towards the cathode!

Andy
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