2015-09-11

TFKE37
Fluorescence Lab

Short guide to GraphPad Prism

1. Open the program
New Table and Graph: Choose XY
Enter/Import data: Choose Enter and plot a single Y value for each point

2. Import your data and name the axis:

X = concentration of DNSA (uM)
Dont forget units!!!
Y = intensity

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3. Analysis:
Mark the data to be analyzed (X and Y column)
! Analyze

Analyze data:
! XY-analyses
! Non-linear regression
! Binding Saturation
! One site Total

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5. Evaluation

Results
! Non-lin fit of Data 1
Note the Kd value and the R2 (R square). The R2 is a measure of the goodness
of fit (should be as close to 1.0 as possible!)

Graphs
! Data 1

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Save the graph as a picture:

! Export
! Export as
! Choose file format (tif, jpg, )
! Export

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The model: One site Total Binding

1

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.
2

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.
3

Model

Y=Bmax*X/(Kd+X) + NS*X + Background

Interpret the parameters

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

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Kd is the equilibrium binding 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.
5

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.