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 <filip.vanpete...@gmail.com> wrote:
From: Filip Van Petegem <filip.vanpete...@gmail.com>
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
