Sandy,
like mentioned previously, sounds like FeS.
record an optical spectrum. Or even better, check whether there is
somebody on the campus running an EPR machine (equipped for helium
temperature measurements)
Maybe a new FeS protein? FeS is not necessarily required as redox
cofactor. It can
According to Pierce TCEP is "more" tolerant of nickel and cobalt. However, TCEP
is inactivated by other metals, namely copper, magnesium, silver and zinc.
Dan
Daniel A. Bonsor,
Boston Biomedical Research Institute,
64 Grove Street,
Watertown,
MA 02472 USA
That's a possibility. Do you know if the same coloration would arise if there
is TCEP instead of DTT or b-ME? I usually have TCEP as the reducing agent.
Thanks Fillip.
Cheers,
Shiva
--- On Fri, 9/24/10, Filip Van Petegem wrote:
From: Filip Van Petegem
Subject: Re: [ccp4bb] protein
>
> I also have a similar observation for proteins purified by Ni-NTA column.
> After concentrating the sample eluted from the Ni-NTA column, I see a
> brownish-yellowish tinge closer to the bottom of the filter with colorless
> buffer on top. This is observed even for non-metal binding and non-FeS
] protein turns brown
To: CCP4BB@JISCMAIL.AC.UK
Date: Friday, September 24, 2010, 4:34 AM
Dear all,
I have purified protein from E.coli. expression system. the protein has been
purified with three independant columns. Now during concentration step using
amicon, the protein shows brown colour. what
Symmons
Cambridge
--- On Fri, 24/9/10, vikrant saa wrote:
From: vikrant saa
Subject: Re: [ccp4bb] protein turns brown
To: CCP4BB@JISCMAIL.AC.UK
Date: Friday, 24 September, 2010, 10:50
sometime it does happen becoz of protein aggregation, or reducing environment.
but it may be your protein color
From: Matthew Bratkowski
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, September 24, 2010 11:38:14 AM
Subject: Re: [ccp4bb] protein turns brown
It could be caused by iron contamination in one of your buffers. We used to
buy
glycerol in a metal canister and metal would leach into the
It could be caused by iron contamination in one of your buffers. We used to
buy glycerol in a metal canister and metal would leach into the glycerol.
Because of this, one protein that I worked with would turn yellow, even at
relatively low concentrations. I did not have this issue when using
glyc
[FeS] clluster ?
Or some metal bound to your protein ?
Is it dark brown or yellowish brown ? What's your protein concentration ? > 25
mg/ml ?
Jürgen
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Instit
Maybe you should give us a hint about the identity of your protein (if you
dare;-)). I'm sure there are folks around who may be able to say whether or
not your protein is supposed to be brown. You can't expect too much help if you
don't provide (m)any details.
Cheers, Bert
On 9/24/10 4:34
Hi Sandy,
If your protein is soluble (i.e. we're not dealing with brown
precipitate), it is possible that the brown color is due to a bound
iron-sulfur cluster. In this case, your protein was always brown, but
you couldn't see the color when it was very dilute. As a quick test,
you could t
Hi Sandy,
Is there any possibility of your protein to contain an iron-sulfur cluster?
Best
Ricardo
Subject: [ccp4bb] protein turns brown
Dear all,
I have purified protein from E.coli. expression system. the protein has been
purified with three independant columns. Now during concentration step using
amicon, the protein shows brown colour. what could be the reason.
best regards and Thanks,
sandy
Dear all,
I have purified protein from E.coli. expression system. the protein has been
purified with three independant columns. Now during concentration step using
amicon, the protein shows brown colour. what could be the reason.
best regards and Thanks,
sandy
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