> > 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
>
to make sure, you could try to spin down this precipitate. then try to
dissolve it in SDS sample buffer and check on PAGE.
> 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?
>
i have seen His-tagged proteins form aggregates before. in that case the
problem probably was the imidazole used to get them off the Ni-column.
i don't know whether a Ni-containing buffer will peel your protein of
the column, and how Ni will affect the protein afterwards though.
how to solve the problem, is quite something different. a suggestion is
to try gelfiltration in 70% formic acid. 70% formic will dissolve almost
everything. gelfiltration in 70% FA is possible.
also, if you could precipitate the protein and then get it into solution
again, you also have an extra purifying step.
HTH,
if only a bit,
jw
