Hi all. I found that reference. :0)
Acta Crystallogr D Biol Crystallogr. 2006 Jul;62(Pt 7):833-42. Epub 2006 Jun 20. Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches. Izaac A, Schall CA, Mueser TC. http://www.ncbi.nlm.nih.gov/pubmed/16790940 They actually used PEG in their test case to force their protein out of solution, but I have used milliQ water successfully in the past. If you are considering using this technique, you probably already know how to precipitate your protein! Good luck! Dave ============================ David C. Briggs PhD Father, Structural Biologist and Sceptic ============================ University of Manchester E-mail: david.c.bri...@manchester.ac.uk ============================ http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB ============================ On 12 August 2010 19:57, Matthew Bratkowski <mab...@cornell.edu> wrote: > 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 > > >
