How to show protein interaction?

D.Lippert lippy at UVic.CA
Thu Nov 28 17:39:16 EST 1996


"Thorsten Schmidt" <Thorsten.Schmidt at rz.ruhr-uni-bochum.de> wrote:

>Dear Reader!

>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?

>Thank you for your answer

>Thorsten Schmidt

If you want the simplest test for presence of dimers, I would have to
say non-denaturing PAGE.  Both structures (monomer and dimer) should
have the same charge to mass ratio, but the dimer, being larger, will
migrate more slowly through the gel due to the sieving properties of
the acrylamide.  This can be done easily in a day without the need for
large complicated equipment.  If you want to prove that the bands you
see are dimers and trimers, run a 2D gel and do SDS-PAGE in the second
dimension.  All of the bands that you see should migrate the same
distance in the second dimension.
I agree that the non-denaturing gel says nothing about molecular
weight, but it WILL separate a dimer from a monomer.  I guess the only
problem would be that it won't tell you how many monomers are in the
higher band (ie. is it a dimer, trimer, tetramer...).  It would be the
fastest and easiest method to get a yes/no answer to the dimerization
question, though.

good luck, Dustin




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