[RASMB] AUC capabilities

[Borries Demeler]

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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