Dear TY,
Typically between 5-10x molar concentration over the protein is enough to ensure binding when the IC50 is uM to low mM. For tighter binding compounds (nM to low uM), 2-5x is sufficient. Whatever you do, when the precipitate occurs DO NOT REMOVE it. I learned to my chagrin that you change all the dynamics of the drop when you do. I ended up with empty crystals until I left the precipitate in place. Think of it this way- Free protein + compound ↔ protein:compound complex + precipitate (mix of protein + compound) If you change the equilibrium by removing the precipitate, you remove the “pressure” on the P:C complex, and it will dissociate to P + C. The precipitate acts as a reserve of protein and compound, thus favoring (or stabilizing) the P:C complex. I set drops up as a slurry frequently, and if I get crystals, they always have the compound bound. Pay attention to the drops if you are screening, because it will be important to note what makes the precipitated solution better (clear drops=solubilizing) or worse (aggregated drops=decreased solubility of your complex). You can also try suspending your compound in LMW PEG’s (200-400 FW) instead of DMSO. Either way, try using DMSO (~20%) or LMW PEG (~30%) depending on your crystallization conditions as a cryoprotectant. Any crystals that grow have some small amount of those agents in them already, so they should be more tolerant of them in higher concentrations. Best of luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Yvonne TAN Yih Wan Sent: Thursday, August 25, 2011 2:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] co-crystallization Hi , I am co-crystallizing a protein with compound and would like to know how much of compound to add to protein solution to start with. I know that the protein binds compound in a 1 to 1 ratio but also noticed that the compound precipitates out of solution when DMSO is diluted off. Where should I start of? A 1 protein :2 compound ratio or more? And what is the best method to determine if the binding is homogeneous (that all protein has got a compound in it)? Any suggestions would help. Thanks TY -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
