Re: [ccp4bb] finicky protein / cell disruption

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: finicky protein
From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU
Date: 2008-03-03

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Subject: protein degraded when setting xtal?
From: Ngo Duc Tri ngoductri {- at -} GMAIL {- dot -} COM
Date: 2008-03-03

Subject: Re: finicky protein / cell disruption
From: Thomas Earnest TNEarnest {- at -} LBL {- dot -} GOV
Date: 2008-03-03

I will second this recommendation to use alternate cell disruption
methods instead of sonication.
Particularly with multi-protein complexes, where the preservation of the
complex from disruption though
crystallization is important, sonication can be quite deleterious.

Even with the French press (or a cell emulsifier), disruption at low
pressures (e.g. three passes
at low pressure) increases the level integrity of full complexes, while
giving good yield.

My recommendation is to throw your sonicator away and use methods that
apply less heat and energy
to your cells and to your biomolecules.

- Th

Thomas Earnest, Ph.D.
Senior Scientist and Group Leader
Structural Proteomics Development Group
Physical Biosciences Division
MS64R0121
Lawrence Berkeley National Laboratory
Berkeley CA 94720

TNEarnest@lbl.gov
510 486 4603

Mads Gabrielsen wrote:
> I am not a big fan of sonication. Try changing your way of disrupting the
> cells.
>
> I have compared sonication vs mechanical stress on several unrelated proteins,
> and for me a good old french press wins every time. If you want to get all
> modern and fancy, a cell disruptor gives similar results.
>
> Cheers,
>
> Mads Gabrielsen
>
>
> [Hide Quoted Text]
>
> On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote:
>
> Hi all
>
> sorry, for offtopic query...
>
> I am trying to purify my protein by Ni-NTA affinity chromatography. After
> sonication as i centrifuge bacterial lysate, soon after 10 min whole
> lysates
> get precipitated during loading on the column and some time it remain
> soluble too. if i get purified through the column without precipitation,
> it
> gets precipitated during dialysis.
> I have tried lot, by chnaging buffers, increasing salt or deacreasing salt
> or no salt at are helpless.
> I do purifiaction in cold room.
>
> can any one suggest some solution?
>
> Thanks in advance.
>
> NSH
>
>
>

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: finicky protein
From: price {- at -} UCHICAGO {- dot -} EDU price {- at -} UCHICAGO {- dot -} EDU
Date: 2008-03-03

Next message:
Subject: protein degraded when setting xtal?
From: Ngo Duc Tri ngoductri {- at -} GMAIL {- dot -} COM
Date: 2008-03-03