protein reduction

Kresten kresten at my-deja.com
Fri Jan 5 04:26:30 EST 2001


> So far I've tried lysis buffer
> containing 10mM DTT combined with 50mM mercaptoethanol but the protein
> still doesn't reduce completely.

Some (many?) proteins does not reduce completely unless you denature
the protein (heat/denaturant). With, say DTT, try using e.g. 50mol DTT
pr. mol disulfide in 8M urea (or 6M Guanidine Hydrochloride) for 30 min
at RT.

Another problem, that I have encountered is that it may be difficult to
detect that the protein is actually completely reduced. The problem is,
that if you use something like Elman to detect free -SH you have to
remove *all* DTT (beta-ME) before this analysis. If your disulfides are
very keen on reforming, some of them might reform during DTT removal
(gel-filtration, dialysis). Low pH and purging your solutions with N2
may help a lot on this.

HTH
Kresten


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