[RASMB] AUC capabilities

Thu Jan 27 14:11:07 PST 2011

Ewa

With regard to Borries' points:

(i) Obviously to get a Kd you need to know the sigma value for your  
monomeric species - working with re-proteins one normally does.know it.  
  And a single run suffices - although I  normally - if for some reason  
good c(s) info was lacking  - would do a short series in c to eliminate  
the effect of inactive bits of dimer.  But one really cannot expect to  
pull out the Kd for a species of unknown sigma value in a polydisperse  
mixture, however you play it.

(ii) simulation immediately shows that with a sigma value of 2, and a  
cell loading concentration of 0.1, you are getting close to  
Beer-Lambert Law in the near-bottom region of the cell. So it's a good  
concentration. Any much higher and you would be obliged to reduce the  
speed to keep within Beer-Lambert across the column. Which loses you  
information.

Regards

Arthur

On Jan 27, 2011, at 17:35, Borries Demeler wrote:

> Ewa,
>
> I think John makes some very good points. The method Arthur recommends
> in my opinion will fail (fitting Kd from a single scan) unless you can
> confidently fix the molecular weight. With the approach John points out
> you can definitely do that, and make sure your sample is in good  
> composition.
> However, you would still need a reasonable absorbance to get that  
> measurement
> reliable, to at least confirm the MW to what is known from sequence.
> I am still skeptical that you will get reliable information given how
> little material you have for testing and how challenging it will be to
> get a good signal to noise at a low enough concentration. 0.1 OD at  
> 220 nm
> is not optimal, especially if you cannot be certain about purity.
>
> -Borries
>
>>
>> Hi Ewa,
>>
>> I just wanted to add that I think your experiment is feasible and  
>> probably a
>> bit easier than implied by some of the others. I remember for example  
>> that
>> Gay-May Wu and her Amgen colleagues reported a Kd of ~30 nM for the  
>> dimeric
>> cytokine stem cell factor, where the monomer mass is only 18.5 kDa,  
>> done via
>> absorbance (280, 230, 215). [Y-R Hsu et al. (1997), J. Biol. Chem.  
>> 272,
>> 6406-6415]. So the higher mass for your system definitely makes it  
>> easier
>> than that published one (which I suspect holds the record for the  
>> lowest
>> product Kd * monomer mass).
>>
>> I don't disagree that it is highly desirable to cover concentrations  
>> 5-10
>> fold above and below the Kd, but doing so is most important when you  
>> aren't
>> sure about the stoichiometry (or it is controversial for some  
>> reason), or
>> when your samples aren't completely pure and homogeneous. But if you  
>> have a
>> nice tight dimer you can use the high concentration data to precisely  
>> define
>> the buoyant mass of the dimer (the end point of the isotherm), and  
>> then with
>> global analysis of multiple experiments you don't need to have all  
>> that much
>> dissociation to monomer in order to define the Kd reasonably well. In  
>> the
>> above study the lowest loading concentrations were 20 micrograms/mL  
>> (1.1
>> micromolar, ~0.02 OD at 230 nm), and the Kd should correspond to  
>> about 0.012
>> OD at 215 nm. Similarly the isothermal titration calorimetry crowd  
>> says they
>> can extract Kd for associations where the concentrations in the  
>> calorimeter
>> are always ~2 orders of magnitude above the Kd.
>>
>> In other words, I am trying to say that the limits of what can or  
>> can't be
>> done are not sharply defined. They depend strongly on the  
>> signal/noise ratio
>> of the measurements, and how well you can define the end-points of  
>> your
>> isotherm (the monomer and dimer buoyant masses for SE). Clearly much  
>> also
>> depends on just how accurately you need to know the Kd. Finally,  
>> feasibility
>> depends too on the quality and stability of your protein samples.  
>> Just a
>> little bit of irreversible aggregate can really throw things off when  
>> you
>> are not able to span the desirable full range of concentrations.  
>> Gay-May had
>> the advantage of having pharmaceutical-grade protein.
>>
>> John
>>
>> -----Original Message-----
>> From: rasmb-bounces at rasmb.bbri.org  
>> [mailto:rasmb-bounces at rasmb.bbri.org] On
>> Behalf Of Ewa Folta-Stogniew
>> Sent: Wednesday, January 26, 2011 6:41 PM
>> To: rasmb at rasmb-email.bbri.org
>> Subject: [RASMB] AUC capabilities
>>
>> Hello,
>>
>> is it possible to determine dimerization constant in the order of 25  
>> nM
>> using AUC?  What will be the minimal amount of protein needed?
>>
>> Ewa
>>
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************************************************************************ 
*******
Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156

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