Re: [ccp4bb] Protein Color

- Protein crystallography

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- Protein purification
- Crystallisation

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- X-ray detectors

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

Previous message:
Subject: Re: Protein Color
From: Joe Cockburn cockburn {- at -} PASTEUR {- dot -} FR
Date: 2008-09-06

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Subject: Re: Protein Color
From: Filip Van Petegem filip {- dot -} vanpetegem {- at -} GMAIL {- dot -} COM
Date: 2008-09-06

Hello Matt,

did you use a nickel (or similar) affinity column in the purification + is
there any beta-mercaptoethanol or DTT in your sample? Nickel can easily
'bleed' off the column and will turn brown when complexed with bME or DTT -
this complex can get quite large and even insoluble if enough is present,
and would therefore concentrate in your sample.

Cheers

Filip

On Sat, Sep 6, 2008 at 4:49 AM, Joe Cockburn wrote:

> Hi Matt,
> I sometimes see a similar thing with my proteins, which definitely don't
> possess metal co-factors or prosthetic groups. I found that gel filtration
> got rid of it - the browny-yellow stuff came out in the void fraction so I
> figured it was aggregated protein. I think it was aggregation via the
> his-tags around traces of copper in my sample, which could explain the
> brown-ish colour.
> What happens if you concentrate the protein in the presence of EDTA?
> Joe
>
> > Hello.
> >
> > I am working with a protein that turns a yellowish-brown color when it is
> > concentrated to around 2 mg/ml or higher in a small volume (a few hundred
> > uL). I was wondering if the protein bound a metal or other prosthetic
> > group that would give it this color? The protein's color somewhat
> > resembles iron binding proteins, but there is no peak in the 400 nm range
> > that would suggest heme, and an iron sulfur cluster is not that likely
> > since there are only five cysteines in the protein. Proteins with
> > structures homologous to the one I am studying bind magnesium, but are
> not
> > know to bind other metals. Any information about what this color might
> > suggest about the protein or how I could analyze possible bound metals or
> > prosthetic groups using only a small amount of protein would be helpful.
> >
> > Matt
> >
>

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpetegem@gmail.com
http://crg.ubc.ca/VanPetegem/

CCP4bb navigation

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

Previous message:
Subject: Re: Protein Color
From: Joe Cockburn cockburn {- at -} PASTEUR {- dot -} FR
Date: 2008-09-06

Next message:
Subject: Re: CNS refinement problem
From: Axel Brunger brunger {- at -} STANFORD {- dot -} EDU
Date: 2008-09-06