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protein precipitates after dialysis


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4 replies to this topic

#1 rtandon

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Posted 16 January 2002 - 08:36 PM

Hi !

I expressed and purified a recombinant protein under denaturing condition, 8M urea using Qiagen Ni-NTA column Everything was OK till the purification step. I got single bands for purified protein. But during dialysis the protein precipitated. I tried to redissolve it using 1%DMSO and also by altering pH but it did not work. How to resolubilize the protein. Please suggest ASAP. Thanks !
rtandon


#2 ragno

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Posted 12 February 2002 - 09:28 AM

Hi,
The critical step in dialysis with 8M urea is 1M to 1,5M urea and if there is precipitate at this molarity, it's finish. You can see qiagen manual about refolding.
Good luck
michel

#3 sean

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Posted 13 March 2002 - 05:47 PM

I had same problem last week.  

What's happening is you are removing the denaturant that kept your proteins in solution.  

When You dialyze denatured proteins you have to make sure that folding is favored as opposed aggregation.
The precipitates you see probably are your proteins aggragated.

Like i said, i had the same thing. I was planning on denaturing my aggregates again with 6M urea or GuHCl. then dilute the proteins sufficiently enough such that foldign is favored. but i haven't tried it yet.


#4 joerg

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Posted 19 July 2002 - 07:09 AM

Maybe you can try to renature your Protein dirctly on the column by stepwise Urea. Since your protein is captured, aggregation should be low.

#5 labslave

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Posted 21 November 2002 - 10:24 AM

I have encounter a similiar problem when dialyzing a my protein in PBS buffer. I added 5 mM beta-mercaptoethanol to all my dialysis buffer. It seems to prevent precipitation during dialysis and due to its low concentration should not cause to many problems with other protocol assays.

Hope that helps!




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