Ni-purification blues again,

Svend Kjaer svend at cajal.mbb.ki.se
Wed Jan 24 11:26:47 EST 2001


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

--
***************************************
Svend Kjær, Ph.D.
Division of Molecular Neurobiology,
Department of Neuroscience
Karolinska Institute
Berzelius väg 1, 3rd floor
171 77 Stockholm
Sweden

Tel: +46-8-7287666
Fax: +46-8-339548

Private phone: +46-8-6409962
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