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
