protein purification and aggregation
jacqg at zpam.dds.nl
Mon Aug 14 15:19:42 EST 2000
> > 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
> 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.
if only a bit,
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