Scatchard Analysis by Flow Cytometry

  • Robert F. Murphy
Part of the Springer Lab Manual book series (SLM)


The goal of the protocols described here is to measure the affinity of a ligand for cell surface receptor(s) and to determine the number of receptors per cell, following the classic method of Scatchard [1]. The preferred method for analysis of ligand binding is to measure ligand bound to cells at equilibrium without removing unbound ligand, since removing unbound ligand allows ligand dissociation to begin and may result in underestimation of the equilibrium value. As first described by Bohn [2] and Steinkamp and Kraemer [3], the small diameters of the sample stream and excitation beam used in most flow cytometers provide excellent discrimination between cell-associated and free ligand. Further discussion of the basis of this discrimination and analysis of the effects of ligand affinity, number of receptor per cell, and sample stream diameter on expected signal (fluorescence from bound ligand) to noise (fluorescence from free ligand) may be found elsewhere [4]. When the equilibrium method is not suitable, the rapid dilution method is frequently an acceptable substitute (see second Procedure).


Free Ligand Core Diameter Equilibrium Method Excitation Beam Scatchard Analysis 
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  1. 1.
    Scatchard G (1949) Ann NY Acad Sci 51: 660CrossRefGoogle Scholar
  2. 2.
    Bohn B (1976) Exp Cell Res 103: 39–46PubMedCrossRefGoogle Scholar
  3. 3.
    Steinkamp JA, Kraemer PM (1979) In: Melamed MR, Mullaney PF, Mendelsohn ML (eds) Flow cytometry and sorting. John Wiley and Sons, New York, pp 497–504Google Scholar
  4. 4.
    Murphy RF (1990) In: Melamed MR, Lindmo T, Mendelsohn ML (eds) Flow cytometry and sorting, Second Edition. Wiley-Liss, Inc, New York, pp 355–366Google Scholar
  5. 5.
    Sipe DM, Murphy RF (1987) Proc Natl Acad Sci USA 84: 7119–7123PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Robert F. Murphy
    • 1
  1. 1.Department of Biological SciencesCarnegie Mellon UniversityPittsburghUSA

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