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
