Hello Frank, thank You for Your response.
> 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.
That is the strange point because I am using the same conditions as other
labs use to purify these proteins. I found different protocols for these
proteins,
and tried them without any success.
>>> By the way, can you _see_ the aggregate? Can you spin them down? Is
> there light scattering?
After thawing I saw a precipitate, therefore I stopped freezing the probes,
but without success. Another observation is that the proteins sometimes get
lost after applying them to a column (gelfiltration, HPLC, PD-10) that means
no
bands on SDS-PAGE. I also noticed an increasing pressure of the column.
Summarized I got two results, either the proteins appeared in the void volume
or they disappeard. Thus my interpretation is that the proteins form aggregats
which either stick to the column or do not interact at all. It is confusing.
But I have no direct proof for the formation of aggregats. What else could
it be?
Thanks, Alexander.
