[RASMB] AUC capabilities

[Borries Demeler]

> 
> 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

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.

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.

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.

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.

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