Ni-NTA/HiTrap-Chelating, urea, the pH, and thiol reagents
wub at gbf-braunschweig.de
Tue Jul 23 10:37:57 EST 1996
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,
Gesellschaft fuer Biotechnologische Forschung
Mascheroder Weg 1
phone: Germany ++49 531-6181 372
FAX: Germany ++49 531-6181 355
e-mail: wub at gbf-braunschweig.de
More information about the Methods