How to show protein interaction?

Leemor Joshua-Tor leemor at CSHL.ORG
Fri Nov 29 10:26:23 EST 1996


Achim Recktenwald wrote:

>Cornelius Krasel 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'd try analytical gel filtration if you have the equipment (i.e. an
>> FPLC/HPLC). Alternatively, you may try analytical ultracentrifugation
>> (I have absolutely no experience with this) or maybe non-denaturing
>> gel electrophoresis.
>>
>> --Cornelius.
>>
>> --
>> /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
>> /* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
>> /* "Science is the game we play with God to find out what His rules are."  */
>
>
>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


Analytical ultracentrifugation is the most precise and direct way to
determine molecular weight and thus oligomeric form, if you can get
access to one. The second choice would be a light scattering experiment
where you are determining the radius of the particle. Both of these
require access to the proper insturmentation. Gel filtration and native
gel-electrophoresis should be always taken with a grain of salt since
they often give misleading results because they are a very indirect
way of getting at the MW of the particles.

-Leemor




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  Leemor Joshua-Tor, Ph.D.
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  Cold Spring Harbor Laboratory           Tel. (516) 367 8821
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