Phosphate seems to be a kind of a 'general, innocuous buffer' that
biologists like so much. It also is a mild solubilizer for many
proteins.
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.
A.G.E.
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 |
