genecutl at mendel.berkeley.edu
Mon Jan 31 07:49:26 EST 1994
In article <genecutl-310194130016 at kos2mac17.berkeley.edu>,
genecutl at mendel.berkeley.edu (gc) wrote:
> I've been involved in the purification of a protein for crystallography
> and have run into something unusual which may cause us problems.
> The protein is expressed in E.coli and run over several columns until
> it is pretty much pure. Sometimes in the E.coli sonicate, the protein
> out as a doublet, with a small amount of the protein running slightly
> higher than the rest. Regardless of the initial amount of this higher
> form, it invariably increases over time, during the purification and
> especially during storage afterwords (at 4 degrees). The abundance of
> the mystery band increases until it is roughly 50% of the total protein.
> We've done some mass spec on the mixture and see only one predominant
> mass, with some minor oxidation products (not enough to account for the
> mystery band).
> Any ideas?
(I should have mentioned that this is not a dimer of the protein. On an
SDS-PAGE the protein runs at around 35kD with the second band right above
that. There are several faint bands at around 60-70kD which I assume to
be unreduced covalent dimers in addition to the unexplained band.)
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