mw132 at mole.bio.cam.ac.uk
Tue Jun 5 11:10:18 EST 2001
. . . I thought there was also some objection from the manufacturer
because Tris can act as an electrophile. However, my chemistry is very
bad and I don't quite know how that can be bad. Mike.
On Mon, 4 Jun 2001, Dr. Artem Evdokimov wrote:
> Phosphate seems to be a kind of a 'general, innocuous buffer' that
> biologists like so much. It also is a mild solubilizer for many
> Buffers that are not recommended would involve substances that can
> chelate metals (things with multiple polar moietiesthat are
> geometrically close, primary amines, etc.). If, for some reason, you
> have high concentrations of transition metals in your lysate then this
> can also influence your purification.
> In practical terms, if you start with phosphate you will be OK (don't
> forget to add some ionic strength, say 200 mM NaCl). MES, HEPES and even
> Tris are also OK in modest concentrations. Avoid sulphides, though some
> BME is also alright. Please take into account the fact that each protein
> is unique and you will very likely run into several proteins that would
> not behave in a predictable manner on Ni-NTA. If your protein binds DNA
> or RNA there is a nonzero chance that it would either not bind at all or
> would bind in an unpredictable manner - dnase/rnase treatment usually
> takes care of that.
> Bacterial cells contain a number of proteins that have high affinity
> towards Ni-NTA resin. The most famous of those proteins is the His-rich
> cis/trans prloine isomerase. it is a 21 kDa protein that you will almost
> always find as a contaminant of your preparation because it has several
> transition-metal-binding sites.
> Hope this helps.
> Lera wrote:
> > Hello!
> > Can anybody tell me why Tris buffer is
> > not a good tool for Ni-NTA resine
> > washing in protein purification?
> > And why phosphate buffer is recomnded?
> > Thanks.
> > Valeria.
> |Dr. Artem Evdokimov Protein Engineering |
> | NCI-Frederick Tel. (301)846-5401 |
> | FAX (301)846-7148 |
> | eudokima at mail.ncifcrf.gov |
> | http://www.ncifcrf.gov/plague |
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