Theresa,
I would suggest then, that you include reducing agent in your buffer (it doesn't appear to be to be there) - TCEP is good, as you can include in right from the start as it doesn't interfere with the IMAC columns. My suspicion is that you are seeing multimerisation through inappropriate di-suphide formation.
Tony.
On 9 Jul 2013, at 08:18, Theresa Hsu <[log in to unmask]>
wrote:
> Dear Tony
>
> Yes, there are four cysteines.
>
> Theresa
>
>
> On Tue, 9 Jul 2013 06:26:19 +0000, Antony Oliver <[log in to unmask]> wrote:
>
>> Theresa,
>>
>> Are there any cysteines in your protein?
>>
>> Tony.
>>
>> On 9 Jul 2013, at 05:01, Theresa Hsu <[log in to unmask]> wrote:
>>
>>> Dear all
>>>
>>> I am working on a 30 kDa membrane protein which forms a functional dimer. The protein is His-tagged at N-terminal. In small scale expression screening from whole cells, there is only a single band on Western blot at 30 kDa. But, after purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large proportion elute near the void volume (8 ml).
>>>
>>> Detail purification
>>>
>>> For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 g. I then checked the supernatant on SDS-PAGE and scale it up for purification.
>>>
>>> For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole, 0.05 % DDM (two times CMC of DDM).
>>>
>>> Is there suggestion to get homogeneous protein?
>>>
>>> Thank you.
>>>
>>> Theresa
>
>
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