How to show protein interaction?

Wulf Dirk Leuschner wdl at gen.mpib-tuebingen.mpg.de
Sat Nov 30 14:11:55 EST 1996


lippy at UVic.CA (D.Lippert) wrote:

>"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

Hi Thorsten,

try crosslinking your proteins! There's a paper describing specific
crosslinking of His-tagged proteins using Ni-ions and
Mg-Monoperoxyophthalic acid (MMPP, try Aldrich). According to the
authors they can crosslink their proteins either on the Ni-NTA-column
or in solution using micromolar (100 - 200) concentrations of both
Ni-acetate and MMPP. If you are using detergents this might present
some problems. I think they tested a few detergents like Triton-X-100
and NP40 up to a concentration of 0.1 % (w/v). I have tried it once
with 1 % CHAPS, but it did not work. Still, this seems to be such a
lovely method, that I have to try it again under sligthly different
conditions...

The reference is:

Fancy, D.A. et al. (1996): New chemistry for the study of multiprotein
complexes: the six-histidine tag as a receptor for a protein
crosslinking reagent. Chemistry & Biology 3, 551-559.







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