Maybe the His Tag is blocked by the folded protein.
Try using 6M Guanidine-HCL and see if it sticks.
Then you will need to find some way of refolding your protein,
if you want to crystallize it.
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
>
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