How to keep recombinant protein stable and soluble?

Anthea Scothern antheascothern at lycos.co.uk
Fri Nov 29 11:13:45 EST 2002


"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
news:J8gA9.8352$tY3.2562905 at twister.neo.rr.com...
>
> 1) Perhaps your protein likes to be in high salt ? Need to check.
> 2) Use DTT in all buffers and freeze the protein ASAP
> 3) do you have activity check to see if the protein that you brought back
> from the dead using reducing agent was actually alive ?
>
> More details would help - what's the pI of the protein, how many
cysteines,
> is it pro- or eukaryotic, etc.
>
> A.G.E.
>

Watch out adding DTT to your buffers for nickel columns - it can reduce the
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.

You can make dialysis a bit more gentle by doing it in steps - eg, 1M salt
down to 750mM, then 750mM down to 500mM etc etc

HTH,

Anthea.





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