Scribd Logo
Upload

Welcome to Scribd!

  • 215 views

    Short Guide To GraphPad Prism

    Uploaded by

    oschlep
    This document provides instructions for analyzing binding data using GraphPad Prism: 1. Import concentration and intensity data into GraphPad Prism and name the axes. 2. Use nonlinear regression analysis to fit a one site - total binding model. This will determine the Kd value and maximum binding (Bmax) without directly measuring nonspecific binding. 3. The model assumes nonspecific binding is proportional to ligand concentration and the Kd is the concentration for half maximum binding. Goodness of fit is indicated by the R2 value.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    Short Guide To GraphPad Prism

    Uploaded by

    oschlep
    215 views6 pages
    This document provides instructions for analyzing binding data using GraphPad Prism: 1. Import concentration and intensity data into GraphPad Prism and name the axes. 2. Use nonlinear regression analysis to fit a one site - total binding model. This will determine the Kd value and maximum binding (Bmax) without directly measuring nonspecific binding. 3. The model assumes nonspecific binding is proportional to ligand concentration and the Kd is the concentration for half maximum binding. Goodness of fit is indicated by the R2 value.

    Original Description:

    Short Guide to GraphPad Prism

    Original Title

    Short Guide to GraphPad Prism

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    This document provides instructions for analyzing binding data using GraphPad Prism: 1. Import concentration and intensity data into GraphPad Prism and name the axes. 2. Use nonlinear regression analysis to fit a one site - total binding model. This will determine the Kd value and maximum binding (Bmax) without directly measuring nonspecific binding. 3. The model assumes nonspecific binding is proportional to ligand concentration and the Kd is the concentration for half maximum binding. Goodness of fit is indicated by the R2 value.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    215 views6 pages

    Short Guide To GraphPad Prism

    Uploaded by

    oschlep
    This document provides instructions for analyzing binding data using GraphPad Prism: 1. Import concentration and intensity data into GraphPad Prism and name the axes. 2. Use nonlinear regression analysis to fit a one site - total binding model. This will determine the Kd value and maximum binding (Bmax) without directly measuring nonspecific binding. 3. The model assumes nonspecific binding is proportional to ligand concentration and the Kd is the concentration for half maximum binding. Goodness of fit is indicated by the R2 value.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 6

    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

    LINKPING UNIVERSITY
    IFM CHEMISTRY

    LINKPING UNIVERSITY
    IFM CHEMSITRY

    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

    LINKPING UNIVERSITY
    IFM CHEMSITRY

    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

    LINKPING UNIVERSITY
    IFM CHEMSITRY

    Save the graph as a picture:


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

    LINKPING UNIVERSITY
    IFM CHEMSITRY

    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.






    LINKPING UNIVERSITY
    IFM CHEMSITRY

    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.

    You might also like