protein purification and aggregation

jw 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
> 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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