Try TCEP as it does not interfere with the NiNTA resin whereas DTT does.
But why do you care if your column is brown or not - can you elute
your protein ?
Are there other metal ions e.g. iron which bind to your resin perhaps ?
Jürgen
On 19 Jun 2009, at 02:25, Kn Ly wrote:
> Hello everyone,
>
> I am trying to purify a 13 KDa membrane protein using Ni NTA. The
> protein is
> solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and
> binds
> very well to the column. However, it also turns the column brownish.
> The protein contains 4 cysteine residues so I suspect that this causes
> cross-linking with other proteins and thus brownish precipitation on
> the
> column. So I included 5 mM beta-ME in my buffer to prevent disulfide
> bond
> formation but this doesn't help. I tried 1 mM DTT and this ruined
> the column.
> Help!! Is there anyway to prevent this brownish problem?
>
> Thanks a lot in advance
> Kien
-
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.me.com/bosch_lab/
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