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- Protein crystallographyMain steps:- Protein purification- Crystallisation Special:- Programs for crystallography- X-ray detectors Basic tutorials:- Chemistry- Protein - Peptide - Amino Acids Xtal community:- CCP4BB |
CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: off topic From: Andy Torelli att29 {- at -} CORNELL {- dot -} EDU Date: 2009-06-19 Hi Kien, You may also want to try to wash your column with elution buffer (without reducing agents), equilibrate it with binding buffer (plus reducing agents) and then bind your protein. In the case of HisTrap columns (a pre-packed, Ni(II) resin column), this procedure is recommended if you wish to use reducing agents. I was told by tech. support that washing with high imidazole elution buffer first (without reducing agents) helps wash out weakly-bound nickel ions that are left over from the charging process. These ions, which are not fully chelated to the resin, are more susceptible to the reducing agents and may undergo the process described by Guenter to result in your brown color. Good luck, -Andy On 6/19/2009 9:19 AM, Guenter Fritz wrote: > 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/ > > CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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