Ni-NTA/HiTrap-Chelating, urea, the pH, and thiol reagents
JM.Ossewaarde at rivm.nl
Wed Jul 24 12:35:10 EST 1996
Did you considered using Talon from Clontech?
I have no experience myself, but according to the documentation up to 30
mM BME might be used.
In article <4t2rkc$1j7 at rzlimes.gbf-braunschweig.de>,
wub at gbf-braunschweig.de (Wulf Blankenfeldt) wrote:
>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
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