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Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
 

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CCP4bb <-- 2008 <-- February 2008 <-- 16 February 2008
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Date: 2008-02-16


Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Jacob Wong jacob {- dot -} j {- dot -} wong {- at -} GMAIL {- dot -} COM
Date: 2008-02-16
Dear all, thank you so much for all the invaluable tips that were delivered
faster than FedEx (and sorry for not being able to reply to individual
emails). My problem was resolved by adding EDTA (to about 5 mM), spinning
down at 15K for 10min to get rid of precipitation (I did have some even with
the help of EDTA), and then a nice HiTrapQ column with Hepes buffer dilution
(many people's favorite it turns out and apparently my protein likes it a
lot since it now concentrates to 10 mg/ml at least). Since I need the
protein for binding assays, I didn't try to add more salt (Q column killer
also), but diluting with 1XPBS (or H2O) doesn't help clear the precipitate -
so it seems that the key things here are imidazole, trace amount of nickel,
and a good buffer.
Thank you so much for all your help, caring and patience. This is a great
community.

Have a nice weekend, -J.J.

On 2/14/08, Jacob Wong wrote:
>
> Dear all,
>
> I just ran into this problem and would like to see if I could get some
> helpful tips before my protein completely crashes out.
>
> I have a protein as 6His fusion and it remained bound to the Ni resin with
> 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM
> (added to 1XPBS). The protein seemed to be highly concentrated in the
> elution and began to get cloudy right away, with more and more precipitation
> produced over a matter of minutes. I felt so helpless, didn't know what to
> do, and then decided to add 5% of glycerol into one of the fractions but
> that made it even more cloudy (ohh no...).
>
>
> While the protein is dying in the tube, do you have some quick remedy for
> me? Thanks very much, -J.J.
>
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