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: [log in to unmask]
http://users.physik.tu-muenchen.de/marius/
phone: +49-(0)89-2891-2550
fax: +49-(0)89-2891-2548
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