Re: [ccp4bb] finicky protein

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

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- Programs for crystallography
- X-ray detectors

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   - Amino Acids

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

Previous message:
Subject: finicky protein
From: Naren sharma narensharma11 {- at -} GMAIL {- dot -} COM
Date: 2008-03-02

Next message:
Subject: Re: finicky protein
From: James Stroud jstroud {- at -} MBI {- dot -} UCLA {- dot -} EDU
Date: 2008-03-03

Subject: Re: finicky protein
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-03-03

If you really tried all sorts of buffers (did you go to extreme pH-values,
very high salt concentrations, tried various additives (beta-ME,
glycerol,...), different salts)
you can still
- try another expression system (insect cells, mammalian)
- see if any modifications might be useful
- try restricted proteolysis - well, you need a little bit of purified
protein for this, but it might be the most promising
- guesstimate a proper subclone from secondary structure prediction of
your
protein.

Tim

--
Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

On Sun, 2 Mar 2008, Naren sharma 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
>

CCP4bb navigation

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

Previous message:
Subject: finicky protein
From: Naren sharma narensharma11 {- at -} GMAIL {- dot -} COM
Date: 2008-03-02

Next message:
Subject: Re: finicky protein
From: James Stroud jstroud {- at -} MBI {- dot -} UCLA {- dot -} EDU
Date: 2008-03-03