Re: [ccp4bb] Protein Color

- Protein crystallography

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

Previous message:
Subject: Re: truncate ignorance
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-09-08

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Subject: SPINE2 Workshop on biophysical characterisation of protein complexes
From: "gohlke, ulrich" ulrich {- dot -} gohlke {- at -} MDC-BERLIN {- dot -} DE
Date: 2008-09-09

Subject: Re: Protein Color
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-09-08

Dear Matt,

Based on the follow-up information that you posted, I would be willing to
bet that the color you observe is caused by various Ni complexes formed with
DTT/BME/etc. Dialysis won't necessarily remove these complexes (especially
if protein is part of the complex) and EDTA may not destroy them either
since affinity of R-S (especially if there's more than one sulfur) for Ni is
typically higher than that of EDTA.

A couple of years ago I had a curious case when depending on fermentation
conditions different metals were incorporated into a protein we were working
with (and at that time we didn't even know it was a metalloprotein!). Metal
content was low - so in dilute form all the protein preps (various mutants
and truncations) were colorless, but upon concentration I was (pleasantly)
surprised by a rainbow of colors - from green (it had Ni), through yellow
(Mn and some Fe), into a nice coral pink (Co with traces of Fe). Some of the
preps remained colorless (later turned out to have Zn).

Good luck,

Artem

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Matthew Alan Bratkowski
Sent: Monday, September 08, 2008 12:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein Color

Thanks for all of the helpful suggestions. In reply to some of the
comments, I did purify by eluting with imidazole from a Ni-NTA column.
The imidazole I used is 99% pure, although I'm not sure whether it is
reagent or molecular biology grade. I also do use DTT in my lysis and
elution buffers, so these could contribute to the color.

After Ni-NTA, I dialyzed the protein into a buffer containing 10 mM Tris,
pH. 7.0, 10% Glycerol, as BME overnight, and then purified it on a
Q-column. I then did a final dialysis in buffer that contained 2 mM DTT
and 5 mM MgCl2 (although the color appears without the MgCl2 as well),
among other reagents. I would think that the two dialysis steps and the
Q-column would remove all of the imidazole. However, I did not run the
protein on a sizing column or use EDTA in any buffers. I could try using
a sizing column, and if that does not remove the color, I could use some
of the techniques suggested to determine if any metals are bound.

Matt

CCP4bb navigation

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

Previous message:
Subject: Re: truncate ignorance
From: Peter Zwart PHZwart {- at -} LBL {- dot -} GOV
Date: 2008-09-08

Next message:
Subject: SPINE2 Workshop on biophysical characterisation of protein complexes
From: "gohlke, ulrich" ulrich {- dot -} gohlke {- at -} MDC-BERLIN {- dot -} DE
Date: 2008-09-09