Re: [ccp4bb] protein turns brown

[Guenter Fritz]

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 have any other function . See e.g. primase from 
eukaryotes (recent structures)
Or in your native (maybe eukarytic) protein is a Zn.  E.coli places 
sometimes a FeS instead of a Zn in the site.
Less likely but further possibility: Ni2+ bound to the protein. If DTT 
is added: Ni2+ -S coordinated is not redox stable and oxidizes to Ni3+ 
(brownish colour).

HTH
Guenter

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
 

Re: [ccp4bb] protein turns brown

[Daniel Bonsor]

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

Re: [ccp4bb] protein turns brown

[shiva bhowmik]

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 turns brown
To: CCP4BB@JISCMAIL.AC.UK
Date: Friday, September 24, 2010, 2:50 PM


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 cluster 
proteins and also for proteins after removal of the His-Tag. This is presumably 
arising due to difference in viscosity where the protein gets concentrated 
closer to the bottom of the filter.



Or more likely:  Nickel bleeding from the column was complexing bME or DTT in 
your sample: Ni-bME forms a tight brown complex.

Filip Van Petegem





  

Re: [ccp4bb] protein turns brown

[Filip Van Petegem]

>
> 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
> cluster proteins and also for proteins after removal of the His-Tag. This is
> presumably arising due to difference in viscosity where the protein gets
> concentrated closer to the bottom of the filter.
>
>
> Or more likely:  Nickel bleeding from the column was complexing bME or DTT
in your sample: Ni-bME forms a tight brown complex.

Filip Van Petegem

Re: [ccp4bb] protein turns brown

[shiva bhowmik]

Hi All,

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 cluster 
proteins and also for proteins after removal of the His-Tag. This is presumably 
arising due to difference in viscosity where the protein gets concentrated 
closer to the bottom of the filter.

Cheers,

Shiva


--- On Fri, 9/24/10, sandeep  wrote:

From: sandeep 
Subject: [ccp4bb] 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 could be the reason.



best regards and Thanks,

sandy 



  

Re: [ccp4bb] protein turns brown

[MARTYN SYMMONS]

and possibly the opposite to reduction which is oxidation - do you have cys 
residues? Perhaps your DTT or TCEP got exhausted? Remember you can add up to 
10mM (not the traditional token 1mM). But remember to neutralize TCEP. Other 
possibility is adventitious metals as these give strong charge transfer bands 
on binding to cys.

So make sure you include EDTA or similar chelator (after metal affinity 
obviously). Oxidation is more likely if your protein unfolds - so try to keep 
it happy with nicely buffered pH and any ligands that might stabilize it.
   Good luck. 
 Martyn
Martyn 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 as well that visible during concentration 
 
 
Vikrant


 
 
 






From: sandeep 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, 24 September, 2010 2:04:54 PM
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 

Re: [ccp4bb] protein turns brown

[Matthew Bratkowski]

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
glycerol from plastic or glass containers though.

Matt

On Fri, Sep 24, 2010 at 9:03 AM, Van Den Berg, Bert <
lambertus.vandenb...@umassmed.edu> wrote:

>  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 AM, "sandeep"  wrote:
>
> 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
>  <
> http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle?>
>
>
>

Re: [ccp4bb] protein turns brown

[Jürgen Bosch]

[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 Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-3655
http://web.mac.com/bosch_lab/

On Sep 24, 2010, at 9:03 AM, Van Den Berg, Bert wrote:

> 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 AM, "sandeep"  wrote:
> 
> 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 
>  
> 
>  

Re: [ccp4bb] protein turns brown

[Van Den Berg, Bert]

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 AM, "sandeep"  wrote:

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
 

Re: [ccp4bb] protein turns brown

[Andy Torelli]

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 take a UV-Vis spectra.  4Fe-4S clusters have a peak around 410 
nm and are distinctly brown in color.


-Andy


===
Andrew T. Torelli Ph.D.
Postdoctoral Associate
Department of Chemistry & Chemical Biology
Baker Laboratory, Cornell University
Ithaca, NY 14853
===

On 9/24/2010 4:34 AM, sandeep wrote:

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

Re: [ccp4bb] protein turns brown

[Ricardo de Pádua]

Hi Sandy,

Is there any possibility of your protein to contain an iron-sulfur cluster?

Best

Ricardo

Re: [ccp4bb] protein turns brown

[vikrant saa]

sometime it does happen becoz of protein aggregation, or reducing environment.
but it may be your protein color as well that visible during concentration 
 
 
Vikrant


 
 
 





From: sandeep 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, 24 September, 2010 2:04:54 PM
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