Ni-NTA/HiTrap-Chelating, urea, the pH, and thiol reagents

[Wulf Blankenfeldt]

Dear everyone,

I am trying to purify a 6xHis-tagged protein from inclusion bodies by
the use of either Ni-NTA from Quiagen or HiTrap-Chelating from
Pharmacia loaded with Ni2+.
My protein likes 8M urea, pH higher than 8 and 25mM DTT (these are the
most successful conditions to solubilize my inclusion bodies; DTT
could possibly be replaced with 100 or more mM beta-mercaptoethanol).
In order not to ruin my Ni-NTA or the Pharmacia column I made some
preliminary experiments checking which reagents Ni2+ can tolerate as I
remembered many metals don't like a basic pH. To my great
disappointment the uncomplexed Ni doesn't tolerate much thiol reagent
and precipitates in urea if pH is higher 7, so I wonder if this
behaviour changes much if it is complexed on the affinity beads. I
also wonder if there is any great difference between the (expensive)
Quiagen material and the Pharmacia stuff.
Any suggestions or experiences to share?

Thanks for reading,


Wulf
-----------------

Wulf Blankenfeldt

Gesellschaft fuer Biotechnologische Forschung
MSF
Mascheroder Weg 1
38124 Braunschweig

Germany

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