Weak binding of His-tags to IMAC

[Han]

"Bean Long" <ben.long at yourfinger.anu.edu.au> wrote in
news:4330f46c$1 at clarion.carno.net.au: 

> Hi all,
> 
> I am having difficulty getting much of my His-tagged protein complex
> to bind IMAC beads (I'm using Talon which has immobilised Co2+).  Most
> of my protein comes out in the washes at pH 8.0 and only a small
> portion comes out when I elute with either imidazole or EDTA.  My
> troubleshooting guide suggests a higher pH might help but 8 is already
> quite high.  I am also using 300 mM NaCl in my wash buffers, could
> this be the problem?  Any ideas appreciated. 
> 
> Cheers,

My his-tagged protein, too, binds relatively weakly to resins.  You just 
have to try different resins and conditions.  I now use histrap_HP columns 
from what is now GE Healthcare, in part because we got the chance to 
purchase an HPLC machine ...  I do not use any imidazole in the binding or 
washing, but still have to really check peak purity (still in the process 
of getting problems solved).

-- 
Best regards
Han 
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