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Dear Indrani,<br><br>
SEC/MALLS with FL detection should complement your AUC with FL detection
in terms of time-dependent changes (if there are any) in dimer
stability. It would be rather unlikely that the dimer falls apart
into partially unfolded monomers that have the same hydrodynamic size as
a dimer. <br><br>
On a well maintained SEC/MALLS system you can go down to 1ug/ml
concentration at teh apex of the eluting peak to <b><u>measure</u></b>
the Mw from SEC/MALLS experiment (and Kd of 60nM can be measured by
SEC/MALLS unless you have very unusual kinetics, but if your Kd changes
with time, it should be picked up by SEC/MALLS as well). In
addition, you can follow dimer dissociation to much smaller
concentrations using FL detection to monitor peak elution position; from
the shift in the peak elution position you can compute dissociation
constant. I do not know it for a fact, but I would guess that FL
detection on HPLC FL detector can probably go to lower concentrations of
protein than FL detection on the AUC system, or at minimum match the
sensitivity you have on AUC. You simply need to assure
that the injection volume to are constant, so all the shift in elution is
due to size changes.<br><br>
how low had you gotten with protein concentrations during your SEC/MALLS
experiments? What are the concentrations in your eluting peak
at the apex of that peak in mg/ml and uM? Have you gotten below you Kd
value for your SEC/MALLS? How big is your monomer? <br><br>
Ewa <br><br>
At 05:13 AM 8/30/2012, Sen Indrani wrote:<br><br>
<blockquote type=cite class=cite cite=""> Dear All,<br>
Thank you Tom, Sophia and Ewa for the replies. I already<br>
had done the SEC_MALS experiment and the mass distribution under the<br>
peak was always homogeneous. The protein is a dimer always as it has
a<br>
coiled coil, but the dimerization is concentration dependent. I also
did<br>
CD of the protein and the melting curve is completing reversible .
The<br>
SEC peak is also almost symmetric with no major trailing or leading<br>
edge. I am limited by the SEC MALS with going down with the<br>
concentration so I have to use FDS_SV in AUC only . The Kd would<br>
defintely be in the nanomolar range and so the question I asked you<br>
before still remains a question. Please could you provide further<br>
insights.<br><br>
Thank you,<br>
Regards<br>
Indrani<br><br>
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To: rasmb@rasmb.bbri.org<br>
Subject: RASMB Digest, Vol 35, Issue 4<br><br>
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<br>
Today's Topics:<br><br>
1. Re: Help needed (Sophia Kenrick)<br>
2. Re: Help needed (Ewa Folta-Stogniew)<br>
3. Re: Help needed (Borries Demeler)<br><br>
<br>
----------------------------------------------------------------------<br>
<br>
Message: 1<br>
Date: Thu, 23 Aug 2012 12:44:56 -0700<br>
From: Sophia Kenrick <skenrick@wyatt.com><br>
To: "rasmb@rasmb.bbri.org" <rasmb@rasmb.bbri.org><br>
Subject: Re: [RASMB] Help needed<br>
Message-ID:<br>
<x-tab> </x-tab><br>
<0E627C11B80B30409FFE2FBB526943ED022D90F369FA@exchange2007.wyatt.com><br>
Content-Type: text/plain; charset="us-ascii"<br><br>
Dear Indrani,<br><br>
As Tom mentioned, definitely look into the possibility of
irreversible<br>
aggregation happening in parallel with your reversibly associating<br>
protein. SEC combined with multi-angle light scattering is an
ideal<br>
method for identifying the molecular weight of each species eluted
from<br>
the column. In the case of irreversible association, the aggregate
peak<br>
should be completely separate from the monomer peak. You can
perform an<br>
SEC-MALS run on an aliquot of your protein after incubating for 1-15<br>
hours to see if the fraction of irreversible aggregate grows with
time.<br><br>
In the case of reversible association, you should see an increase in
Mw<br>
across a single peak as compared to the expected monomer molar mass
for<br>
your protein. For a reversibly associating protein, the main peak
in an<br>
SEC chromatogram will shift to higher Mw as you inject a higher<br>
concentration on the column (or larger volume of the same<br>
concentration). Depending on the kinetics of the reaction, you may
be<br>
able to measure a higher molar mass at the very apex of the peak
(where<br>
the concentration is highest) as compared to the leading or trailing<br>
edge where the concentration is lower. However, often you get
a<br>
constant, average Mw across the peak that changes as a function of
the<br>
amount of protein that was loaded on the column. You can get a<br>
semi-quantitative estimate of the KD from this type of
experiment.<br><br>
A more accurate quantification of oligomerization KD by light
scattering<br>
would be performed in batch. Composition gradient static light<br>
scattering (CG-SLS or CG-MALS) involves measuring the light
scattering<br>
signal of different concentrations of an unfractionated
macromolecular<br>
solution. As complexes form, the weight average molar mass of
the<br>
solution should increase. The change in light scattering (Mw) as
a<br>
function of composition can then be fit to an appropriate
association<br>
model to yield affinity and stoichiometry. Below is a link to a
book<br>
chapter reviewing this technique, and the references listed in it may
be<br>
useful to you.<br><br>
<a href="http://www.intechopen.com/books/protein-interactions/characterization-of" eudora="autourl">
http://www.intechopen.com/books/protein-interactions/characterization-of</a>
<br>
-protein-protein-interactions-via-static-and-dynamic-light-scattering<br>
<br>
Best regards,<br>
Sophia<br><br>
Sophia Kenrick, Ph.D. | Application Development Engineer Wyatt<br>
Technology Corporation 6300 Hollister Avenue | Santa Barbara, CA<br>
93117-3253<br>
Phone: (805) 681-9009 x297 | Fax: (805) 681-0123<br>
Web:
<a href="http://www.wyatt.com/" eudora="autourl">www.wyatt.com</a>
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http://www.wyatt.com</a>> | E-mail:<br>
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------------------------------<br><br>
Message: 2<br>
Date: Thu, 23 Aug 2012 16:21:49 -0400<br>
From: Ewa Folta-Stogniew <ef55@email.med.yale.edu><br>
To: Sophia Kenrick
<skenrick@wyatt.com>,<x-tab>
</x-tab>
"rasmb@rasmb.bbri.org"<br>
<x-tab> </x-tab>
<rasmb@rasmb.bbri.org>,<x-tab> </x-tab>Sen Indrani
<Indrani.Sen@psi.ch><br>
Subject: Re: [RASMB] Help needed<br>
Message-ID:
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------------------------------<br><br>
Message: 3<br>
Date: Thu, 23 Aug 2012 17:56:49 -0500<br>
From: Borries Demeler <demeler@biochem.uthscsa.edu><br>
To: Ewa Folta-Stogniew <ef55@email.med.yale.edu><br>
Cc: RASMB List <rasmb@server1.bbri.org><br>
Subject: Re: [RASMB] Help needed<br>
Message-ID: <20120823225649.GA17138@biochem.uthscsa.edu><br>
Content-Type: text/plain; charset=us-ascii<br><br>
On Thu, Aug 23, 2012 at 04:21:49PM -0400, Ewa Folta-Stogniew
wrote:<br><br>
> _______________________________________________<br>
> At 03:44 PM 8/23/2012, Sophia Kenrick wrote:<br>
><br>
> Depending on the kinetics of the reaction, you may be
able to<br>
measure a<br>
> higher molar mass at the very apex of the peak (where
the<br>
concentration is<br>
> highest) as compared to the leading or trailing edge
where the<br>
> concentration is lower. However, often you get a
constant, average<br>
Mw<br>
> across the peak that changes as a function of the amount
of protein<br>
that<br>
> was loaded on the column.<br>
><br>
> and:<br>
><br>
> You can get a semi-quantitative estimate of the K[D]
from this type<br>
of<br>
> experiment.<br>
><br>
> the last sentence is not true; depending on the kinetics
of the<br>
> association, you can get quantitative estimate of Kd
for<br>
self-association-<br>
> you just need to prove that this is the case.<br>
><br>
> Wouldn't the kinetics of self-association be a concern
for use of<br>
sed.<br>
> velocity for Kd determination as well?<br>
><br>
> Ewa<br><br>
It's not really a concern when all you want is a Kd. For purposes of<br>
determining rate constants, the question is whether the reaction is
too<br>
fast compared to the time scale of the velocity experiment. Whether
it<br>
is or not depends on the size of the molecule and the speed with
which<br>
you are running. If the reaction is too fast, you simply
cannot<br>
determine the rate constant of the reaction, but the Kd will still
be<br>
measurable. In the extreme case of non-interaction, you will simply
get<br>
two boundaries corresponding to monomer and oligomer. (for kd<br>
calculation you need molar concentrations, and you need to keep in
mind<br>
that the dimer absorbs twice as much as the monomer)<br><br>
In the other extreme, when the reaction is faster than the time scale
of<br>
the vcelocity experiment, you get a reaction boundary, which changes<br>
gradually from monomer towards oligomer as you go up in the<br>
concentration gradient. In my experience for a typical protein the
upper<br>
limit of a k_off you can measure in a velocity experiment at 60krpm is
a<br>
rather slow speed of about 10^-4/sec. For all reactions, the Kd
should<br>
still be easily measurable as long as you have enough signal from
both<br>
the monomer and the oligomer.<br><br>
-b.<br><br>
<br>
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