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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Roger Rowlett
Sent: Thursday, August 25, 2011 6:11 PM
To: CCP4BB@JISCMAIL.AC.UK
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: rrowl...@colgate.edu
