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Re: [ccp4bb] His tag does not bind.
 

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CCP4bb <-- 2007 <-- October 2007 <-- 11 October 2007
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Subject: b-factor sharpening and FFT/CAD
From: Roni Gordon rgordon {- at -} UHNRESEARCH {- dot -} CA
Date: 2007-10-11 		Next message:
Subject: Re: His tag does not bind.
From: hong yu honghyu {- at -} HOTMAIL {- dot -} COM
Date: 2007-10-11


Subject: Re: His tag does not bind.
From: Kendall Nettles knettles {- at -} SCRIPPS {- dot -} EDU
Date: 2007-10-11
We have found that our His-MBP fusion doesnąt bind well after we cut off the
protein of interest, and are trying to remove it. We have to use very low
salt, cold temp, and slow loading rates. You might also try batch instead of
column loading. We have also had good luck adding 1-2M urea to uncut
His-MBP-protein fusions that show poor binding to the Qiagen Ni-NTA.
Kendall


On 10/10/07 9:12 PM, "changrui lu" wrote:

> Dear all,
>
> I am trying to express a 150 kd protein in E coli. I have it in two
> constructs, one with pmal-his and other with only his tag at N terminus. The
> full length protein can be detected both by sds and western using anti-his
> (190kd and 150kd respectively) but strangely neither binds to his-column very
> well. The majority of the full length comes through the column either at
> loading step or low salt wash step. The major species that gets trapped and
> eluted is the mbp-his truncation (~40kd). Some, though very little, full
> length protein did make it out the his column. The pmal-his construct does not
> bind amylose resin any better with majority flows right through. All
> purification are carried out under standard conditions as mentioned in the
> manuals. The protein is soluble and does not precipitate in the columns. I
> appreciate and ideas or explanations.
>
> Thanks in advance.
>
> Ray
> Cornell Univerisity
>





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