Hi YT- We normally prepare our ligand stocks in DMSO and add this to the protein in 3-fold molar excess. The majority of our ligands are quite insoluble and precipitate when the DMSO concentration decreases upon addition to the protein....... so I am not surprised that you are seeing this. If your compound does not bind your protein tightly, you might consider using a 5-fold molar excess of ligand.
Some proteins crash out if the protein concentration in high when you add the ligand. For those situations, we complex the ligand with dilute protein (1-2 mg/ml), and then concentrate this for crystallization trials. I have had proteins where we had to complex the dilute protein with ligand, and then let it sit overnight at 4C before we concentrated the protein. We normally incubate the protein+ligand at 4C for 1-3 hours for binding before we set up the crystallization experiments. Another scenario might be addition of ligand to the protein followed by incubation at room temp for ~1hr. Then centrifuge at 4C, keep protein at 4C and set up your trays. Hope this helps! annie From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Yvonne TAN Yih Wan Sent: Thursday, August 25, 2011 2:03 AM To: [email protected] 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
