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If you do the calculations, you will find that you need a FREE ligand concentration of >10 * Kd to get >90% occupancy of the binding site. If you have e.g. a ligand with a Kd of 100 nM, you would need a free ligand concentration of 1 µM. However, a solution of 10 mg/ml of a protein of 30 kDa, has a protein concentration of 333 µM, so in theory you should have a total ligand concentration (free + bound) of 334 µM. In pratice, some of the ligand may have been degraded during (prolonged) storaged, or the compound may not be as pure as the chemist would have wished it to be, so it is wise to use a safety margin of at least two. We normally use 1-2 mM compound to be on the safe side. Having too much ligand usually does not hurt, except that you use more compound, but with too little ligand you end up with an empty binding site and you will have to repeat the experiment with more ligand. Best, Herman ________________________________ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Roger Rowlett Sent: Thursday, August 25, 2011 6:11 PM To: [log in to unmask] Subject: [ccp4bb] Fwd: Re: [ccp4bb] co-crystallization Successful complexation depends on the concentration of protein, ligand, and the Kd of the protein-ligand complex. For Kd>>[protein], you will probably require [ligand] > 10 x Kd. As Kd approaches [protein], slightly superstoichiometric quantities will be sufficient for full occupancy. For Kd < [protein], stoichiometric quantities of ligand will suffice. Basically you need a [ligand] that puts near saturation on the binding isotherm. Cheers. On 8/25/2011 2:03 AM, Yvonne TAN Yih Wan wrote: 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 -- ________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask]