Equation: Two sites - Fit logIC50

Introduction

You can determine the equilibrium dissociation constant of an unlabelled ligand by measuring its competition for radioligand binding. This model assumes that there are two classes of sites with identical affinity for the radioligand, but different affinities for the competitor.

Step by step

Create an XY data table. Enter the logarithm of the concentration of the unlabeled compound into X, and binding into Y. If you have several experimental conditions, place the first into column A, the second into column B, etc. Use subcolumns to enter replicates.

From the data table, click Analyze, choose nonlinear regression, choose the panel of Competition Binding equations, and choose Two sites - Fit logIC50.

Model

Span=Top-Bottom

Section1=Span*FractionHi/(1+10^((X-LogIC50HI)))

Section2=Span* (1-FractionHi)/(1+10^((X-LogIC50Lo)))

Y=Bottom + Section1 +Section2

Interpret the parameters

Top and Bottom are plateaus in the units of Y axis.

FractionHi is the fraction of all the sites that have high affinity for the competitor.

logIC50Hi and logIC50Lo are the logarithms of the two IC50 values.

Notes

This model fits the two IC50 values of the unlabelled ligand. It does not report the two Ki values. The Ki values depend on the IC50s, the concentration of radioligand, and its Kd for binding. You can fit the Ki values directly using a different equation.

This analysis assumes that the binding is reversible and at equilibrium. It also assumes that the labeled and unlabeled ligands compete for the same binding sites.