[RASMB] AUC capabilities

Ewa Folta-Stogniew ef55 at email.med.yale.edu
Wed Jan 26 20:29:18 PST 2011


At 10:39 PM 1/26/2011, you wrote:
> >
> > 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
> >
>
>Hi Ewa,
>
>the answer to your question is: it depends on several factors...
>
>1. what optical system do you plan to use - there are choices:
>absorbance, Rayleigh interference, fluorescence emission

absorbance or interference


>2. If you use absorbance, there is a question about the extinction
>coefficient and the wavelength. Depending on extinction, 25 nM may
>be well within reach at several detection wavelengths.

A280(0.1%)=0.85  (mg-1mL cm-1)

>3. Related to (2) is the question of size - the larger the protein,
>presumably the larger the extinction coefficient (more trp, tyr residues
>at 280 nm, more peptide backbone absorbance at lambda <= 230 nm). Ditto
>for Rayleigh interference - the larger the protein, the more refraction
>you will see and 25 nM could be well within range.


59 kDa monomer


>4. If you consider using the fluorescence detector, then 25 nM label
>of the right excitation/emission should be well within the range of
>detection.

no fluorescence detection available


>5. in order to get accurate Kds you would want to measure ideally
>at concentrations above and below the Kd to get good signal
>of the monomer and the dimer, so multiple concentrations measured
>either in velocity or equilibrium mode and globally fitted should
>give you the desired information. Related to that is that the
>Kd needs to be in the range where you are measuring. If your Kd is
>100 micromolar and you are measuring at 25 nM and below, your answer
>is going to be quite unreliable. At the same token, measuring at
>100 uM when your Kd is at 20 nM will not give you the desired result.

Assuming Kd of 25 nM and the extinction coefficient as above- can one 
determine Kd for dimerization from 50 micrograms of protein total (25 
micrograms preferred)?

If this is theoretically feasible, does anybody know of any published 
AUC results for similarly low Kd?

thanks,

Ewa


>Your question about how much protein is needed at a minimum depends
>also on the questions I posed above. If you provide additional
>information we may be able to tell you more specifically for your sample.
>
>Regards, -borries




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