Help w/ Ni column protein purification

Koen De Smet k.desmet at nospam.ic.ac.uk
Tue Jan 20 08:32:06 EST 1998


Keith wrote:
> 
> In our lab, we are using a hexa-his tagged protein overexpressed in a pET21b
> vector (IPTG inducible).  I have tried to use the Novagen instructions for
> binding the tagged protein to a Nickel column.  The protein doesn't seem to
> stick well and comes out during elution with WASH buffer (60mM imidazole)
> rather than the STRIP (100mM EDTA). Both are in 0.5M NaCl, 20mM Tris-HCl.
> 

I have never used the Novagen columns, only the Qiagen ones. 
I have also noticed that my proteins don't stick very well when I use Tris buffers, but 
they do when I use phosphate buffer (as recommended by QIAGEN). They come off with 50 mM 
imidazole in Tris, but around 150 mM in phosphate. Their manual states that Tris (and 
HEPES and MOPS) have secondary or tertiary amines which will reduce the Ni ions. 

Koen De Smet


Koen


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