How to keep recombinant protein stable and soluble?
AEVDOKIMOZ at cinci.rr.com
Fri Nov 29 13:01:09 EST 2002
"Anthea Scothern" <antheascothern at lycos.co.uk> wrote in message
news:as83ns$2p3i$1 at godfrey.mcc.ac.uk...
> Watch out adding DTT to your buffers for nickel columns - it can reduce
> nickel on the column to a depressing brown precipitate and all will be
> lost - I usually add 1mM 2-mercaptoethanol to all my buffers - too low a
> concentration to reduce the nickel, but enough to maintain the activity of
> the protein.
There's a whole thread on the dangers of DTT in IMAC chromatography, if you
look it up on google. I just assumed that the poster understands that
particular problem :)
One comment, though - being a picky person that I am - DTT is not reducing
Ni++, it just binds Ni++ very tightly and indeed creates the unholy brown
precipitate that is impossible to fully remove from the column. BME (any
thiols) in IMAC buffers will also lower the capacity and the affinity of the
columns, and will eventually destroy the column - just not as fast.
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