(no subject)

Nick Theodorakis via methods%40net.bio.net (by nick.theodorakis from gmail.com)
Thu Jul 16 16:01:44 EST 2009


On Jul 16, 8:29 am, "Vicky Cook" <vc... from wfubmc.edu> wrote:
> Hello,
>
> I am trying to produce TEV protease, and I have a few questions.  First,
> why is DTT added to this enzyme?

I don't know about this enzyme specifically, but in general for
enzymes that contain free sulfhydryl groups often DTT or some other
reductant is included in its storage or reaction buffer to endure that
it doesn't get oxidized.

> I think it has to do with its being a
> cysteine protease.  Second, I am observing strands or strings in my
> dialysis bag, when I dialyze eluate from His bind column (in 20mM
> Tris-HCl pH 7.9, 500mM NaCl, 1M imidazole) into STB (10mM Tris-HCl pH
> 8.0, 150mM NaCl, 1mM EDTA, 0.02% sod. azide, 5mM DTT).  I think it may
> have to do with either the salt concentration or the DTT?  

It's not uncommon for a protein to precipitate when the salt
concentration is changed.

>BTW, is it
> imidizole or imidazole?  I have seen it written both ways.  I think they
> are two different chemicals, but I am trained as a biologist, not too
> good with chemistry....  :)
>

Imidazole. Azoles are a class of compounds with five member rings
containing nitrogen:

<http://en.wikipedia.org/wiki/Azole>

Nick


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