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Hi, can you perform a thermal shift assay ? Google or it or find some references in a posting not too long ago perhaps 6 months or less. If so you can check your protein with various "additives" and see which ones stabilize your protein. What happens if you dialyse your sample instead of concentrating it to get rid of the imidazole ? Jürgen Daniel Jin wrote: > Hi there, > > > > Sorry for the off topic questions. We need your feedback. > > > > We are expressing a rat protein in insect cells. It is expressed as a > secreted protein with an N-terminal 6xHis tag. We can get about 4 mg > of it from 1L culture and everything looked quite normal at the very > beginning (at 4C). When I changed the buffer to HBS using centricon to > get rid of imidazole (@ 4C), I noticed that it took a long time to > concentrate and I saw some ppt. However, when I took some of it (at > about 1.2 mg/ml) and kept them at room temperature, the solution > turned cloudy in a few minutes. I tried to change the pH by diluted in > 1M stock of different buffers (pH 4.5-8.5), change the NaCl > concentration, and add 10% glycerol, but it still crashed out at RT. > However, it seems OK, I hope, when kept on ice. I am wondering whether > any of you had a similar experience before. It is not a problem for us > to do everything at 4 degree. I just worry that it may indicate > something wrong with this protein. The protein should be stable since > it has been shaking at 27C for four days… > > > > Many thanks. > > > > Best, > > Chen__ > > -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch