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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