Dear Tony Yes, there are four cysteines.
Theresa On Tue, 9 Jul 2013 06:26:19 +0000, Antony Oliver <antony.oli...@sussex.ac.uk> wrote: >Theresa, > >Are there any cysteines in your protein? > >Tony. > >On 9 Jul 2013, at 05:01, Theresa Hsu <theresah...@live.com> 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
