Sandra & Thomas Haselhorst wrote:
> Hi there,
>> I am trying to express a ~20 kDa protein in E.coli LB and M9 minimal
> media. I have got good expression and I want to purify this
> recombinant protein with a metal affinity (TALON) column. But my
> protein doesn't bind to the matrix at all. I only find my protein in
> the through flow and elution of the metal column with 150 mM Imidazole
> results in several other bands. But not my protein. Is there anything
> wrong with the matrix, vector etc. ?? I am not sure where to start to
> solve this problem ...
>As Duncan suggests, the tag could be buried. It could also bind to the resin
so weakly that your binding conditions are not appropriate. I have a His
tagged protein that will bind to a Nickel column in the absense of
imidazole, but the inclusion of even 5 mM imidazole in the binding buffer
greatly reduces my yield. I elute the protein in 50 mM imidazole, rather
than the 300 mM that the Qiagen manual suggests. This protein will also not
bind the resin in batch but must flow through the column slowly, on the
order of 3 mLs/hour. Perhaps your protein is similarly finicky.
