Jürgen Bosch schrieb:
> 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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Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
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