Hi Matt, Check out the following paper and some screens available commercially based on these: Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug 26. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Jancarik J, Pufan R, Hong C, Kim SH, Kim R.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Dec 1;61(Pt 12):1035-8. Epub 2005 Nov 5. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Collins B, Stevens RC, Page R. The first one is available as a kit (JBS solubility screen): http://www.jenabioscience.com/cms/en/1/catalog/739//Search http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptSolukit It appears that you have not yet set up screens on this protein. I think it is worth trying: a) Setting up crystallization screens under dilute conditions. Some proteins crystallize under such conditions. b) Set up screens at the higher concentration immediately after concentrating before precipitation starts occurring. c) Try adding some detergent and I think glycerol should help to improve solubility You can also check out Hampton's PCT Pre-Crystallization Test. Thanks, -Debanu. From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Matthew Bratkowski Sent: Thursday, August 12, 2010 11:57 AM To: [email protected] Subject: [ccp4bb] Suggestions for Reducing Protein Precipitation 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
