GraphPad Curve Fitting Guide

Equation: Two sites -- Specific binding only

Equation: Two sites -- Specific binding only

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Equation: Two sites -- Specific binding only

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Introduction

In a saturation binding experiment, you vary the concentration of radioligand and measure binding. The goal is to determine the Kd (ligand concentration that binds to half the receptor sites at equilibrium) and Bmax (maximum number of binding sites) of both kinds of receptors.

The ligand binds not only to receptors sites, but also to nonspecific sites. There are three approaches to dealing with nonspecific binding.

Subtract off the nonspecific, and analyze only the specific binding. Read on for this approach.

Analyze the total binding only, inferring the amount of nonspecific binding from the shape of the total binding curve. This approach doesn't work well when there are two classes of receptors.

Globally analyze the total and nonspecific binding at one time. Learn more.

Step by step

Create an XY data table. Enter radioligand concentration into X, and specific binding into Y. If you have several experimental conditions, place the first into column A, the second into column B, etc.

An alternative approach would be to enter total binding into column A, and nonspecific into column B. Then use the Remove Baseline analysis to subtract column B from column A, creating a new results table with the specific binding.

From the table of specific binding, click Analyze, choose nonlinear regression, choose the panel of Saturation Binding equations, and choose Two sites -- Specific binding.

Model

Site1=BmaxHi*X/(KdHi+X)

Site2=BmaxLo*X/(KdLo+X)

Y=Site1 + Site2

 

Interpret the parameters

BmaxHi and BmaxLo are the maximum specific bindings to the two sites in the same units as Y.

KdHi and KdLo are the equilibrium dissociation constants, in the same units as X. It is the radioligand concentration needed to achieve a half-maximum binding at equilibrium

Scatchard plots of two site binding

The left panel below shows binding of a radioligand to two independent binding sites present in equal concentrations, but with a tenfold difference in Kd . The two individual curves are shown as dotted and dashed curves. When you do the experiment, you can't observe the individual components, but observe the sum, which is shown as a solid curve. Note that this curve is not obviously biphasic.

The right panel shows the same data plotted on a Scatchard plot. The binding to each receptor is shown as a straight line (dotted, or dashed). The total binding, plotted on a Scatchard plot, is curved. Note that the two lines that represent binding to each type of receptor are NOT the asymptotes of the curve.

To plot the two straight lines that correspond to the nonlinear regression fit, create a new data table that defines the two lines as shown below, using Bmax and Kd values determined by nonlinear regression.

X

A

B

0

Bmax1/Kd1

 

Bmax1

0

 

0

 

Bmax2/Kd2

Bmax2

0

 

Go to the graph of the Scatchard transformed data and drag the new table to that graph. Use the Format Graph dialog to plot the two data sets from the table using connecting lines but no symbols.