GraphPad Curve Fitting Guide

Equation: One site -- Total binding

Equation: One site -- Total binding

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Equation: One site -- Total binding

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Introduction

You don't have to measure nonspecific binding directly. Instead, you can determine Bmax and Kd by fitting only total binding by assuming that the amount of nonspecific binding is proportional to the concentration of radioligand.

Step by step

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

Use any convenient units for X and Y. The Kd will be reported in the same units as X, and the Bmax will be reported in the same units as Y.

From the table of total binding, click Analyze, choose nonlinear regression, choose the panel of Saturation Binding equations, and choose One site -- Total.

Consider constraining the parameter Background to a constant value of zero. This is the measured 'binding' when there is no radioligand binding added, so represents the counter background, if there is any.

Model

Y=Bmax*X/(Kd+X) + NS*X + Background

 

Interpret the parameters

Bmax is the maximum specific binding in the same units as Y.

Kd is the equilibrium dissociation constant, in the same units as X. It is the radioligand concentration needed to achieve a half-maximum binding at equilibrium.

NS is the slope of nonspecific binding in Y units divided by X units.

Background is the amount of nonspecific binding with no added radioligand. This represents counter background. If your counter automatically subtracts off the background signal, you can constrain Background to a constant value of zero.

Notes

This analysis assumes that only a small fraction of radioligand binds, which means that the concentration you added is virtually identical to the free concentration. If you can't make this assumption, use an alternative analysis.