Fitting total binding data to determine Bmax and Kd

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Fitting total binding data to determine Bmax and Kd

In many systems, nonspecific binding is proportional to radioligand concentration over the concentration range used in binding studies. If this is true for your system, you can determine Kd and Bmax by fitting total binding only. Nonspecific binding is determined via curve fitting of total binding, and is not determined experimentally.

To fit total binding, use this user-defined equation:

Specific = X*Bmax /(Kd + X)
Nonspecific = NS*X
Y = Specific + Nonspecific

Variable	Comment
X	Radioligand concentration. Usually expressed in nM or pM.
Y	Total binding in cpm, sites/cell, fmol/mg or some other units
Bmax	The maximum specific binding to be fit. A reasonable initial value might be that Bmax equals 0.5 times YMAX .
Kd	The equilibrium dissociation constant. A reasonable initial value might be 0.2*XMAX
NS	Multiply NS times X to calculate nonspecific binding at that concentration of radioligand. A reasonable rule for initial value is to set NS equal to 0.2 * (YMAX-YMIN)/(XMAX-XMIN).

Checklist for saturation binding