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/
