Hi. I am working with a protein that has difficulties staying in solution when concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0, 5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute, but seems to precipitate onto an anion exchange column during loading, as noticed by brown spots on the column and a reduction in yield after the run. Despite these issues, I have been able to concentrate the protein to about 18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT. However, after sitting at 4 C for a few hours, I again notice precipitate in the concentrated stock. I figure that I will probably just work with more diluted protein next time, but was concerned that my protein was not stable enough in this buffer to use for crystallization.
I am seeking advice on finding a better buffer for the protein. Can anyone suggest whether making subtle buffer changes, such as using HEPES instead of Tris or KCl instead of NaCl would be advantageous? Additionally, I was interested I find out what concentration of glycerol or NaCl could still be added for the protein to still be usable for crystallization, as well as any additives that will prevent precipitation but not interfere with crystallization. Also, could someone suggest a good way to test protein stability in various buffers on a small scale before deciding on conditions for a large batch. Thanks, Matt
