Hi Careina,
Since alternative methods are being suggested...
ITC can be good for quantitating a monomer-dimer equilibrium by
diluting dimers out from a concentrated solution (which obviously
favours the dimer) - and presuming a reasonable Kon/Koff.
Alan Cooper has kindly figured out the data fitting for the rest of us:
http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf
I think Alan was using a VP-ITC when he was doing this stuff. Lower
volumes - and presumably concentrations if the KD is small enough -
are feasible in an ITC200. The protein is recoverable anyway.
All the best,
Will.
On 14 February 2014 12:40, Williams, John Charles <[log in to unmask]> wrote:
> Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this.
> ________________________________________
> From: CCP4 bulletin board [[log in to unmask]] On Behalf Of FOOS Nicolas [[log in to unmask]]
> Sent: Friday, February 14, 2014 12:25 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] KD of dimerization, off topic
>
> I agree with Dave, and i suggest one more method to estimate Kd,
> The intrinsic fluorescence of proteins thanks to the aromatic chain side.
> Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure.
>
> ________________________________________
> De : CCP4 bulletin board [[log in to unmask]] de la part de David Briggs [[log in to unmask]]
> Envoyé : vendredi 14 février 2014 09:13
> À : [log in to unmask]
> Objet : Re: [ccp4bb] KD of dimerization, off topic
>
> Hi Careina,
>
> I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged.
>
> Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation.
>
> Hth,
>
> Dave
>
> On 14 Feb 2014 08:03, "Careina Edgooms" <[log in to unmask]<mailto:[log in to unmask]>> wrote:
> Dear CCP4 board
>
> I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column?
> I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient)
>
> Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization
> Thanks and sorry for off topic question
> Careina
>
>
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