Theresa, Are there any cysteines in your protein?
Tony. On 9 Jul 2013, at 05:01, Theresa Hsu <[email protected]> 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
