[RASMB] AUC reproducibility

[pjgb at mrc-lmb.cam.ac.uk]

Dear Mircea,

I do not usually fit average mass over the whole sample, but rather start 
by plotting the weight average apparent mass (for typically 41 datum 
points) against concentration.  Once I am confident about any concentration 
dependence, I then go on to fit the raw data with the relevant model, 
deriving the monomer mass and also a dissociation constant/non-ideality B 
factor etc.
In my experience these have been repeatable within a few percent, although 
the absolute value for the mass may be out, depending upon how one has 
obtained v-bar - but then I am often working with awkward systems such as 
detergent solubilised membrane proteins where the protein may be binding 
several times its own mass of detergent.
In other words, I have not found reproduceablilty a problem, unless I have 
something else happening such as protein degradation or irreversible 
aggregation, in which case the failure to repeat can be one of the first 
indications that all is not well.

Yours,

Jo

--On Monday, April 4, 2005 1:38 pm +0200 "Rasa, M.V" <m.rasa at tue.nl> wrote:

>
> Dear Jo,
>
> Thank you for the answer. I tried the same thing but I could not
> understand
> why I have such dispersed values. What should I expect from XL-A
> regarding the average molar mass determined from ideal equilibrium
> profiles?
>
> Sincerely,
>
> Mircea.
>
>
>
>> We regularly repeat analyses of aggregating macromolecular samples,
>> frequently from a different preparation, and find that the
>> results are
>> reproduceable.  (Indeed, we regard this as a requirement for
>> publication of
>> any analysis.)
>> If they are not, I want to know why not, as it typically
>> means that some,
>> previously unknown, factor is uncontrolled.



P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296