[RASMB] AUC capabilities

[John Philo]

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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