[RASMB] AUC capabilities

John Philo jphilo at mailway.com
Thu Jan 27 09:01:42 PST 2011


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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