[RASMB] AUC capabilities

[Ewa Folta-Stogniew]

Thanks.  I only consider AUC.   I already had measured the Kd by 
light scattering, but would like to confirm what I had gotten if I 
can figure out how.

SPR is not a method of choice for oligomerization studies due to 
non-homogeneous analyte (i.e. the protein will be different mixtures 
of monomer and dimer when the dilutions are made).

Ewa

At 08:16 AM 1/27/2011, Leech, AP wrote:
>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
>Technology Facility               *  Molecular Interactions Laboratory
>Department of Biology (Area 15)   *  Tel   : +44 (0)1904 328723
>University of York                *  Fax   : +44 (0)1904 328804
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