From: Theresa Hsu
Date: 16 April 2012 08:31
Dear all
I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT?
Thank you.
Theresa
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From: Tim Gruene
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 cleavage did not work?
Cheers,
Tim
- --
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From: Dima Klenchin
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 up the amount of the enzyme. But it's almost certain that most proteins with S-S bridges will be perfectly happy at low reducer concentration (0.2 mM TCEP, 1 mM DTT or something along these lines; particularly if there is more than one bridge - mass action is a powerful thing and, e.g., IgG has no problem at 10 mM DTT). So I wouldn't worry about it - just add enough TEV under conditions that make your protein happy.
Dima
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From: Florian Brückner
----------
From: Jason Forse
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 else's suggestion to add more TEV. That's worked for me as well, as long as the TEV's relatively fresh and there isn't too much reducing agent introduced along with it.
Jason
----------
From: Tim Fenn
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.
HTH,
-Tim
Date: 16 April 2012 08:31
Dear all
I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT?
Thank you.
Theresa
----------
From: Tim Gruene
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 cleavage did not work?
Cheers,
Tim
----------
From: Dima Klenchin
I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT?
Dima
----------
From: Florian Brückner
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:
-------------------------------------------------------------
Dr. Florian Brückner
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From: Jason Forse
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 else's suggestion to add more TEV. That's worked for me as well, as long as the TEV's relatively fresh and there isn't too much reducing agent introduced along with it.
Jason
----------
From: Tim Fenn
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.
HTH,
-Tim
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