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

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
Subject: brom- / iodo- DNA source?
From: JINJIN ZHANG zhang {- dot -} 508 {- at -} OSU {- dot -} EDU
Date: 2008-02-19

Next message:
Subject: Re: Check the conformation of one important amino acid
From: Li Zhijie zhijie {- dot -} li {- at -} UTORONTO {- dot -} CA
Date: 2008-02-19

Subject: Re: rescuing crashing-out protein eluted from Nickel column
From: Charlie Bond Charles {- dot -} Bond {- at -} UWA {- dot -} EDU {- dot -} AU
Date: 2008-02-19

Hi Jacob,
Two points which I don't think others have mentioned:

Have you checked the pH of your imidazole (or pHed the solution before
elution)? It may be that the imidazole has driven up your pH, causing
protein to crash out.

You can add various things to fraction tubes prior to elution so that
the protein gets into the appropriate buffer ASAP (e.g. more buffer for
dilution or 1M NaCl ...)

Cheers,
Charlie

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.

--
Charlie Bond
Professorial Fellow
University of Western Australia
School of Biomedical, Biomolecular and Chemical Sciences
M310
35 Stirling Highway
Crawley WA 6009
Australia
Charles.Bond@uwa.edu.au
+61 8 6488 4406

CCP4bb navigation

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

Previous message:
Subject: brom- / iodo- DNA source?
From: JINJIN ZHANG zhang {- dot -} 508 {- at -} OSU {- dot -} EDU
Date: 2008-02-19

Next message:
Subject: Re: Check the conformation of one important amino acid
From: Li Zhijie zhijie {- dot -} li {- at -} UTORONTO {- dot -} CA
Date: 2008-02-19