[RASMB] Possible for a complex to break apart from force of sedimentation?

[Steven Shire]

Hi Ronald,
  I was following the suggestions and answers to your question. If all the
subunits in your complex are disulfide linked I doubt that forces and
pressures in the AUC run will disrupt those covalent linkages. You may want
to verify that in the complex all the subunits are linked via disulfides. A
simple test would be an SDS PAGE or CE SDS non gel sieving to see if there
are any lower molecular weight entities. A separate experiment using DTT
can determine if the entire oligomeric complex breaks down to 60kDa
subunits.
       Steve

On Wed, Dec 16, 2015 at 11:32 AM, Toth IV, Ronald T <Toth.Ronald at ku.edu>
wrote:

> Thanks everyone, I really appreciate the replies, I have several thing to
> try and think about :-)​
>
>
> - Ronald Toth
>
> Research Associate
> Macromolecule and Vaccine Stabilization Center
> University of Kansas
> ------------------------------
> *From:* RASMB <rasmb-bounces at list.rasmb.org> on behalf of Toth IV, Ronald
> T <Toth.Ronald at ku.edu>
> *Sent:* Monday, December 14, 2015 4:15 PM
> *To:* rasmb at rasmb.org
> *Cc:* Kumru, Ozan S
> *Subject:* [RASMB] Possible for a complex to break apart from force of
> sedimentation?
>
>
> Hi all! We’re looking at a ~60 kDa protein that forms a ~ 1 MDa oligomeric
> disulfide linked complex. By SEC and DLS the sample is very monodisperse at
> about what we would expect the MDa complex to look like. However by AUC we
> see a very heterogeneous distribution between 5s and 30s. I was wondering
> if anyone else had observed a system where a complex had broken apart from
> the force of sedimentation?
>
>
> Thanks in advance!   ​
>
>
>
> - Ronald Toth
>
> Research Associate
> Macromolecule and Vaccine Stabilization Center
> University of Kansas
>
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>
>


-- 
Steven J. Shire, Ph.D.
Consultant
Genentech, Inc.
1 DNA Way
S. San Francisco, CA 94080
650 257 3558 (VOICE)
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