DTT "inhibitor" ?

Emir REMOVEem at unforgettable.com
Tue Dec 4 11:15:22 EST 2001


"BL" <benny at mrc-lmb.cam.ac.uk> wrote in message
news:benny-0412011125350001 at macx33.mrc-lmb.cam.ac.uk...
> Is there any reagent that will specifically stop the -S-S- breaking (i.e.
> reducing) ability of dithiothreitol, without affecting proteins otherwise?
> I am trying to break the -S-S- bonds in some soluble proteins by 8M Urea
> and 50mM DTT. However I need to get rid of the DTT completely before the
> next step as a linker I use contains an -S-S- bond. The only way I can
> think of is to use dialysis to get rid of the DTT. Is there a more
> complete way?
DTT does not attach to the protein, and just reduces the S-S bond. So,
dialysis (bag or column, I would prefer column) will be the most appropriate
way to get rid of DTT.
>
> On a related note, say, when I have dialysed the DTT against just 8M urea,
> how quickly will the -S-S- bonds re-form, considering that it's in a
> denaturing environment?
Flush all your buffers with inert gas to remove oxigen, and try to stay as
anaerobic as possible -- disulfides will never re-form. Even in the aerobic
environment the bond will not form quickly, but depends on the protein. Just
to give you an idea, one would have to stir a short peptide  (say 1-10 kDa,
containing 2 cisteines, 1 mg/ml solution) for 3-4 days in the air to oxidize
it.

If your system tolerates DMSO, you can add this chemical to promote further
oxidating conditions.

Emir





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