Hi Kien,
DTT coordinates very nicely the Ni(II) on the column. However the
DTT-Ni(II) is prone to oxidation which gives almost immediately Ni(III)
which causes the brownish colour. As Jürgen pointed out TCEP chelates
only weakly Ni(II) and is a better choice. Also beta-mercapto-ethanol
does not bind Ni(II) so tightly, i.e causes less trouble. Regarding your
4 Cys residues, there might be some redox going on, but not necessarily.
The Cys SGs are often inside in an hydrophobic environment.
You can do easily the Ni(II) column WITHOUT any reductant in the
buffer. Simply add DTT after the Ni(II) column.
You can also check the oxidation state of your Cys by good old
biochemistry
(http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides).
In order to avoid oxidation of Cys or Met on the Ni(II) column, strip
your Ni(II) and charge your IMAC column with Zn(II) instead of Ni(II).
Zn(II) is redox inert.
HTH
Guenter
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/
--
***********************************
Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz
e-mail: guenter.fr...@uni-konstanz.de
Tel.: +49-(0)7531 88 3687
Fax: +49-(0)7531 88 2966
