Ni-purification blues again,

[Michael Witty]

Could the problem be that the his tag is buried?  You could try using the
"denatured" method of purification described in the Qiagen manual for
Ni-NTA.  Good luck, Mike.

> Dear Netters,
> 
> I have expressed substantial amounts of a receptor using a stable
> cell-line set-up.
> The receptor is his-tagged, which ideally should allow for facile
> purification using Ni-NTA beads added to concentrated serum-free
> conditioned medium.
> However, this is not the case. Each time I try to purify the protein, I
> get some back, but the majority passed through. From the same
> preparation, I have now tried three times and each time the same. The
> majority is in the flow-through and some is eluted specifically.
> I used a 50 mM Tris/150 mM NaCl, 1mM Imidazole buffer and wondered
> whether there are any suggestions to how to solve this problem - could
> the buffer be the reason - or could there be any compound in the
> conditioned medium, which competes with the his-tagged protein for
> binding ?
> 
> Any input appreciated,
> 
> Svend
> 
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> Svend Kjær, Ph.D.
> Division of Molecular Neurobiology,
> Department of Neuroscience
> Karolinska Institute
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