[RASMB] AUC capabilities

[Ewa Folta-Stogniew]

Hi John,

thanks for your input.

Your last comment: " 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."

nails my main problem... not such luck here... as I said: ~ 20 ug per 
prep at best... I also was concerned about the aggregation; the 
monomeric form of the protein tends to aggregate (I can see it 
clearly by LS) .

Also "Clearly much also depends on just how accurately you need to 
know the Kd. "  I suppose what we have is sufficient for this 
project, but it would be great to have the Kd determined from two 
different approaches if it was not a full research project by itself...

What I did was simple "service for fee" from a core lab that I pushed 
a bit beyond routine recommendations... the first sample I got was 5 
ug of protein to test what we can do by SEC/LS.

Ewa

At 12:01 PM 1/27/2011, John Philo wrote:
>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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