protein purification and aggregation

Frank Fuerst fant.1 at gmx.net
Fri Aug 4 12:03:10 EST 2000


Alexander wrote:
> 
> But in all cases I got the same phenomenon, the proteins did not bind to
> anything, they always came down in the void volume. [...]I tried urea in
> different concentrations, but in all cases I got no specific binding to
> any material.
>     In my opinion, it is due to the assembly of huge complexes. But I do
> not why I am not able to break them down. [...]
>     Therefore I read a lot of postings in this newsgroup and saw, that
> this is a common problem, but I could not find a specific solution for
> it despite of playing around with all the parameters and waiting for
> good luck. But I have the impression that there is something basically
> going wrong. Despite of all things I tried I did not even get a hint of
> success.

Well, if you really have aggregated protein, playing around with solvent
parameters won't help you much, since aggregates usually are rather
stable (kinetically). You would have to use considerable concentrations
of urea or guanidinium chloride (5-8 M) to solubilize the protein. With
all that denaturant in the solution I fear you won't have much luck with
doing chromatography, except affinity chromatography, so you have to let
them refold first.
And this might be the next problem: You'll probably have trouble finding
conditions where they refold, since they even aggregate during your
first purification step.

By the way, can you _see_ the aggregate? Can you spin them down? Is
there light scattering?

Greetings, Frank




>     Thus I hope that there is somebody out there, who has an idea of
> what is going wrong (sorry, I can not summerize all the methods, it is
> too much), so that I do not go crazy and depressed by my stupidity.
> 
> Thank you for reading this long posting,
> 
> Alexander Drung
> 
> ---

-- 
Da geht von "alles muss uebers Administrationstool laufen" bis "am
Besten deinstalliert man das Administrationstool sofort nach der In-
stallation". Suse Linux bringt es nach Ansicht vieler irgendwie fertig, 
in beide Kategorien gleichzeitig zu fallen 8-) [MUFTI in dcoum]






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