[RASMB] AUC capabilities

[Leech, AP]

Hi All,

Eva, if you put all 50 ug into 120 uL for an equilibrium experiment,
this would be ~0.4 mg/ml, or 7 uM, giving an approx absorbance of
0.4 at 280 nm. If your Kd is right, this would practically all be
dimer. To get down to sub-Kd concentrations, as Borries says, the
absorbances would be impractically low.

If you put it all into 400 uL for a velocity experiment, the initial
concentration would be 0.125 mg/ml, 2.1 uM, absorbance ~0.13 at 280nm.
This is well above the Kd also, I will leave someone with more
experience to say if dissociation would affect the boundary detectably,
I suspect not.

It might be worth considering SPR. If you can attach one monomer to
the chip surface without compromising dimerisation (practically I think
you would attach the dimer and look for dissociation; there might be
a problem with both halves of the dimer attaching), then wait for
dissociation and flow various concentrations over the surface. The
amounts are more in the ball park for what you have (say 10 ug for
immobilisation and a few ug for each trial of free protein) though
it's still not a lot! and you need to allow for control experiments
for non-specific binding etc.

Best regards,

Andrew

On 27/01/2011 04:29, Ewa Folta-Stogniew wrote:
> 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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-- 
Dr Andrew Leech                   *  Laboratory Head
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