protein purification and aggregation

Randy rldurren at bchserver.bch.ncsu.edu
Thu Aug 17 07:51:16 EST 2000


I haven't got the whole string of this message, but after the His-band
column, why not dialyze into a salt/DTT environment (or another reducing
agent of your choice). Then when you run the imidazole gradient, just add
the reducing agent in the imidazole buffers. It may be worth a try.
Randy Durren


"jw" <jacqg at zpam.dds.nl> wrote in message
news:1efavai.1qc9m081c73t9wN at stol-117-240.uva.studentennet.nl...
>
> > > 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
>







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