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Dear Theresa,
I was not aware you need DTT for TEV protease activity. People do
on-column digestion, and as far as I remember, a Ni-column would turn
really uglily brown of you used DTT on those columns.
Have you tried to leave out DTT and the
I want to digest a tagged protein with TEV protease, it has disulfide
bridges. Is there any way of doing cleavage without DTT?
Yes, no problem. TEV is slowly inactivated oxidation of the active site
cysteine but that's about it. If you absolutely must have no reducer during
cleavage, simply
I have used 5 mM beta-Mercaptoethanol, which is a weaker reducing agent than
DTT, and that keeps TEV happy as well.
Cheers
Florian
Am 16.04.2012 um 09:31 schrieb Theresa Hsu:
Dear all
I want to digest a tagged protein with TEV protease, it has disulfide
bridges. Is there any way of
I've run into the same problem, and found David Waugh's FAQ to be a great
resource:
http://mcl1.ncifcrf.gov/waugh_tech.html
They use a 3mM buffer of 10:1 reduced:oxidized glutathione. I've tried that and
it cleaves my protein without reducing reducing the disulfide bridges.
I'll second someone
Also keep in mind that many of the purchased TEVs are formulated with
some reducing agent (e.g. AcTEV comes in a buffer with 5mM DTT, if I
recall correctly). So unless the enzyme is buffer exchanged
beforehand, there will be some reducing agent introduced alongside it,
depending on the dilution.
