Analyzing Binding Data

Harvey J. Motulsky1, Richard R. Neubig2

1 GraphPad Software, La Jolla, California, 2 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.5
DOI:  10.1002/0471142301.ns0705s52
Online Posting Date:  July, 2010
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Measuring the rate and extent of radioligand binding provides information on the number of binding sites, and their affinity and accessibility of these binding sites for various drugs. This unit explains how to design and analyze such experiments. Curr. Protoc. Neurosci. 52:7.5.1‐7.5.65. © 2010 by John Wiley & Sons, Inc.

Keywords: binding; radioligand; radioligand binding; Scatchard plot; receptor binding; competitive binding curve; IC50; Kd; Bmax; nonlinear regression; curve fitting; fluorescence

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Table of Contents

  • Introduction
  • Binding Theory
  • Saturation Binding Experiments
  • Competitive Binding Experiments
  • Kinetic Binding Experiments
  • Two Binding Sites
  • Agonist Binding
  • Use of Fluorescence or Other Spectroscopic Methods in Binding Experiments
  • Basic Protocol 1: Fluorescence Saturation Binding of BODIPY FL‐GTPγS to GαO
  • Analyzing Data Using Nonlinear Regression
  • Evaluating Results of Nonlinear Regression
  • Comparing Treatment Groups
  • Calculations with Radioactivity
  • Analyzing Data with GraphPad Prism
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Fluorescence Saturation Binding of BODIPY FL‐GTPγS to GαO

  • Ligand (BODIPY‐FL‐GTPγS; Invitrogen) stock solution: 10 µl of 100 µM frozen in 1 mM DTT
  • G protein (Gα o) stock solution: 20 µM snap frozen in HED buffer
  • Binding buffer: HED buffer (see below) containing 10 mM MgCl 2
  • HED buffer: 50 mM HEPES, pH 8, containing 1 mM EDTA and 1 mM DTT
  • Competing ligand (e.g., 100 µM GTPγS)
  • Costar 3915 black 96‐well microplate
  • Fluorescence plate reader
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Literature Cited

Literature Cited
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   Gordon, G.W., Berry, G., Liang, X.H., Levine, B., and Herman, B. 1998. Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy. Biophys. J. 74:2702‐2713.
   Kenakin, T. 2006. A Pharmacology Primer, Second Edition: Theory, Applications, and Methods. Academic Press, San Diego.
   Lakowicz, J.R. 2006. Principles of Fluorescence Spectroscopy, 3rd ed., Springer, New York.
   Limbird, L.E. 2004. Cell Surface Receptors: A Short Course in Theory and Methods, 3rd ed., Springer, New York.
   McEwen, D.P., Gee, K.R., Kang, H.C., and Neubig, R.R. 2001. Fluorescent BODIPY‐GTP analogs: Real‐time measurement of nucleotide binding to G proteins. Anal. Biochem. 291:109‐117.
   Motulsky, H.J. and Mahan, L.C. 1984. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25:1‐9.
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   Neubig, R.R. and Cohen, J.B. 1979. Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: Stoichiometry and ligand interactions. Biochemistry 18:5464‐5475.
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   Roman, D.L., Talbot, J.N., Roof, R.A., Sunahara, R.K., Traynor, J.R., and Neubig, R.R. 2007. Identification of small‐molecule inhibitors of RGS4 using a high‐throughput flow cytometry protein interaction assay. Mol Pharmacol. 71:169‐175.
   Rosenthal, H.E. 1967. A graphic method for the determination and presentation of binding parameters in complex systems. Anal. Biochem. 20:525‐532.
   Swillens, S. 1995. Interpretation of binding curves obtained with high receptor concentrations: Practical aid for computer analysis. Mol. Pharmacol. 47:1197‐1203.
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