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Dear Theresa,
you might also try changing the resin. I have worked on a protein which
would no bind to Ni-NTA (Qiagen) at all but could greatly be purified
when using Ni-IDA (then Pharmacia) instead.
The protein expressed to about 60mg/l LB in E. coli and was well folded.
Best wishes,
Tim
On 01/15/2012 07:23 PM, Theresa H. Hsu wrote:
> Hi all
>
> I have a His-tagged soluble protein (8 His residues added to 90 kDa protein) that do not bind to IMAC column based on flowthrough showing up with Western blott. Do you have suggestions to improve the binding?
>
> Binding condition is 50 mM Tris-HCl 8.0, 300 mM NaCl, 10 mM imidazole pH to 8.0.
>
> Thank you.
>
> Theresa
>
- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
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