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Re: [ccp4bb] Off topic: His-tag purification
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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999
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Subject: Re: Off topic: His-tag purification
From: "Edward A {- dot -} Berry" BerryE {- at -} UPSTATE {- dot -} EDU
Date: 2012-01-15		Next message:
Subject: Re: Off topic: His-tag purification
From: Cécile Breyton Cecile {- dot -} Breyton {- at -} IBS {- dot -} FR
Date: 2012-01-15


Subject: Re: Off topic: His-tag purification
From: Artem Evdokimov artem {- dot -} evdokimov {- at -} GMAIL {- dot -} COM
Date: 2012-01-15

Your protein is either misfolded/aggregated or the his tag is chewed
off/never translated. You could try detergents etc. To improve the state of
the protein but I would also doubl check sequence for early termination
(assuming cterm) or protelysis.
Artem
On Jan 15, 2012 12:23 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
>
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