[RASMB] AUC capabilities

[Ewa Folta-Stogniew]

Hello all,

as pointed by a few already, the anisotropy changes are probably my 
best route at this point, but it would require labeling and wouldn't 
the aggregation of the monomeric form a problem as well?   We may try 
intrinsic fluorescence, but I was told it more likely would not be 
strong enough FL signal (here again, I am not an FL expert, but I can 
ask others at Yale and simply try it with next prep).

Thank you all who responded,

Ewa

At 01:08 PM 1/27/2011, John Philo wrote:
>Ewa, just to clarify, my response was focused on instrumental/technical
>feasibility and was ignoring the problem of sample supply. I agree with
>Borries that my approach requires good, high signal/noise data at
>concentrations >= ~100-fold above Kd to pin down the dimer buoyant mass.
>Thus you would need a couple of channels (120 microliters each) at loading
>concentrations giving ~0.3 OD at 280 nm.
>
>So that would require multiple 20 microgram preps, and unfortunately when
>the yields are that low I would bet that the purity isn't too great.
>
>Another thought is that sometimes you get lucky and the association will
>change the intrinsic fluorescence, and if so that might give you a handle on
>confirming the Kd with only low amounts of protein needed.
>
>John
>
>-----Original Message-----
>From: Borries Demeler [mailto:demeler at biochem.uthscsa.edu]
>Sent: Thursday, January 27, 2011 9:35 AM
>To: jphilo at mailway.com
>Cc: 'Ewa Folta-Stogniew'; rasmb at server1.bbri.org
>Subject: Re: [RASMB] AUC capabilities
>
>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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