HIS TAG - Metal affinity column

Duncan Clark junk at hgmp.mrc.ac.uk
Fri Mar 21 07:50:41 EST 2003


Historians believe that in newspost 
<da9848bd.0303201841.19f44949 at posting.google.com> on Thu, 20 Mar 2003, 
Sandra & Thomas Haselhorst <haselhorst_2000 at yahoo.de> penned the 
following literary masterpiece:
> 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 ...

It could simply be that the His tag is folded up in such a way in your 
protein that it is inaccessible to the column.

Is it N-terminal or C-terminal tagged?

Is your protein a multimer?

What buffer are you using to load the protein on the column?

Duncan

-- 
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.



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