making disulphide bonds to aid refolding

James Bassuk bassuk at
Fri Jan 18 02:49:16 EST 2002

   Each protein is different -- some like a quick refold while others
enjoy a slow refolding process.  While your HisProtein is bound to the
NiNTA column, wash the contaminants away at pH 6, thenraise the pH to
8.5ish (needed for oxidation), and run a reverse gradient of urea from 8
to 0 over 2 hrs.  You might need to include a redox potential, e.g. 50:1
ox:red glutathione (1mM:0.02mM). Then do your pH 6.0 wash again, then
elute with 5.3 and finally 4.5.  Neutralize your fractions with Tris (use
filter paper to get to 7ish), take an aliquot for non-reducing gels.
Good luck,

On Wed, 9 Jan 2002, Michael Witty wrote:

> Dear All,
>         my particular protein at the moment, a phage coat protein,
> contains disulphides which cause a ladder of monomers: one-mers right up
> to sizes that precipitate.  I can break them with 10mM DTT, but when I try
> to refold the protein by removing DTT and 8M urea (protein bound to
> Ni-NTA, to prevent agregation) I get back my ladder of multimers.  I
> suspect that the "good" disulphides are slow to form and the "bad"
> disulphides are fast to form.
>      My solution would be to speed up disulphide formation while protein
> is bound to Ni-NTA: is there a convenient reagent for this?  I have read
> discussion of DMSO on this newsgroup before.  Is this the only commonly
> used reagent?  What concentration do people use?
>      Also, should I expect a protein to snap back into shape upon removing
> 8M urea or is it a time consuming process?
>      TIA for all comments.  Regards, Mike.

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