I would try the following: 1. Remove the His tag. 2. If you need to get rid of the imidazole I would try adding ammonium sulfate. 3. Is your protein proline- and/or rich in aromatic residues? This may explain your need for imidazole and solubility.
> 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 daysÂ… > > Many thanks. > > Best, > Chen > > >
