Abstract
A fluorescence polarization assay can be used to evaluate the strength of a protein-protein interaction. A green fluorescent protein variant is fused to one of the protein partners. The formation of a complex is then deduced from an increase in fluorescence polarization, and the equilibrium dissociation constant of the complex is determined in a homogeneous aqueous environment. The assay is demonstrated by using the interaction of the S-protein and S-peptide fragments of ribonuclease A as a case study.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110:2685–2708
Smith DS, Eremin SA (2008) Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules. Anal Bioanal Chem 391:1499–1507
Owicki JC (2000) Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J Biomol Screen 5:297–306
Royer CA, Scarlata SF (2008) Fluorescence approaches to quantifying biomolecular interactions. Methods Enzymol 450:79–106
Park S-H, Raines RT (1997) Green fluorescent protein as a signal for protein–protein interactions. Protein Sci 6:2344–2349
Park SH, Raines RT (2000) Green fluorescent protein chimeras to probe protein–protein interactions. Methods Enzymol 328:251–261
Park SH, Raines RT (2004) Fluorescence polarization assay to quantify protein–protein interactions. Methods Mol Biol 261:161–166
Jameson DM, Sawyer WH (1995) Fluorescence anisotropy applied to biomolecular interactions. Methods Enzymol 246:283–300
Ormö M, Cubitt AB, Kallio K et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 237:1392–1395
Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251
Richards FM, Vithayathil PJ (1959) The preparation of subtilisin modified ribonuclease and separation of the peptide and protein components. J Biol Chem 234:1459–1465
Watkins RW, Arnold U, Raines RT (2011) Ribonuclease S redux. Chem Commun 47:973–975
Richards FM (1958) On the enzymic activity of subtilisin-modified ribonuclease. Proc Natl Acad Sci U S A 44:162–166
Raines RT (1998) Ribonuclease A. Chem Rev 98:1045–1065
Kim J-S, Raines RT (1993) Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci 2:348–356
Schreier AA, Baldwin RL (1977) Mechanism of dissociation of S-peptide from ribonuclease S. Biochemistry 16:4203–4209
Baldwin RL (1996) How Hofmeister ion interactions affect protein stability. Biophys J 71:2056–2063
Connelly PR, Varadarajan R, Sturtevant JM et al (1990) Thermodynamics of protein–peptide interactions in the ribonuclease S system studied by titration calorimetry. Biochemistry 29:6108–6114
Pédelacq JD, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Raines, R.T. (2015). Fluorescence Polarization Assay to Quantify Protein-Protein Interactions: An Update. In: Meyerkord, C., Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 1278. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2425-7_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2425-7_19
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2424-0
Online ISBN: 978-1-4939-2425-7
eBook Packages: Springer Protocols