determination of binding constant of protein-ligand
damartin at bioslave.uio.no
Wed Feb 14 08:22:24 EST 1996
In article <4fs0g7$agm at netserver.univ-lille1.fr>,
Philippe Lefebvre <plefebvre at pop.univ-lille2.fr> wrote:
-->Whatever method you are using to separate bound from free ligand, you have
-->ligand concentrations from 1/10 Kd to 5 Kd, keeping in mind that the data
-->at very low ligand concentrations will be critical for determining the Kd.
Good quality data all round is important. For Kd the lower end of the range is
important. For Bmax the higher end of the range is important.
Better to stretch the range to more than 1 order of magnitude (10X or 1/10X
Kd), collect lots of data with many repeats and see what it looks like.
-->have to plot the data according to Scatchard.
Scatchard is a linear representation of the binding equation
Bound = ([ligand] x Bmax) / (Kd + [ligand])
This looks very much like the michaelis menten equation.
It is better to fit the equation directly to the data rather than enhance
errors in one dimension by transforming to the scatchard plot. There are
plenty of free programs that will do curve fitting for you. Try the EMBL
(the shape of the curve and how well it fits will also help you to determine
where the deficiencies in your method are)
hope this helps.
* David Martin, PhD - Post-Doctoral Research Fellow *
* Atherosclerosis and Thrombosis research group *
* Biotechnologisenteret i Oslo *
* Gaustadalleen 21 (Postboks 1125 Blindern) *
* N-0316 Oslo *
* Norway *
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* email: david.martin at biotek.uio.no *
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