Exactly, solving the equation is necessary to estimate the required concentration!!!! Set up the eqn., use large volume (1mL) for soaking solution and small volume (1microL) for the crystal: therefore C(free) ~ C(total), simplify, solve for bound ligand in crystal. Thats all. And I can tell you: stoichiometry plus a few times the Kd is usually NOT sufficient. Just calculate it.
M > In those old chemical kinetics courses it was explicitly or > implicitly clear that [I] refers to I(free), not I(total). > The way assays are usually run, [E]<<K(I), so to a good > approximation the amount of bound ligand is negligible. > > The way we set up crystallization, [E] is often mM > while K(I) is uM or below, and you had better consider > concentration AND total amount. You will never get > decent occupancy if you add 1 uM ligand to 1 mM protein, > even if the K(I) is 10 nM! On the other hand if you > ignore K(I) and add stoichiometric ligand, dissociation > of a few time K(I) to satisfy the free concentration > will not hurt occupancy significantly. > If you add 2x stoichiometric to > be safe, you may also fill some very low affinity > (100 uM) site. Use stoichiometric pus a few x Kd, > or solve quadratic equation to see how much is > really required. > > Marius Schmidt wrote: > >> think about your old chemical kinetics courses. >> what counts is concentration and not amount. >> >> M >> >> >>>Dear Marius and others, >>>here I would like to comment: The problem with soaking is often not >>>so much the concentration of the ligand, but the amount of ligand >>>needed to fill all binding sites in the crystal. A typical crystal >>>contains about 20 mM protein so one has to add the equivalent amount >>>of ligand. On the other hand, the concentration UNBOUND inhibitor, >>>asuming a 1:1 protein-ligand complex, need only to be in the order of >>>10-100 times the Kd (90-99% occupancy). Ways to overcome this is to >>>soak in a large volume of mother liquor (containing a large amount of >>>ligand) or to add solid ligand to the mother liquor and let the >>>ligand slowly dissolve and diffuse into the crystal. >>> >>>Herman >>> PD Dr. habil. Marius Schmidt Physikdepartment E17 Technische Universitaet Muenchen James Franck Strasse 85747 Garching/Germany email: [EMAIL PROTECTED] http://users.physik.tu-muenchen.de/marius/ phone: +49-(0)89-2891-2550 fax: +49-(0)89-2891-2548
