UNIT 20.8 Spectroscopic Methods for the Determination of Protein Interactions

  1. Yvonne Groemping1,
  2. Nadja Hellmann2

Published Online: 1 MAR 2005

DOI: 10.1002/0471140864.ps2008s39

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Groemping, Y. and Hellmann, N. 2005. Spectroscopic Methods for the Determination of Protein Interactions. Current Protocols in Protein Science. 39:20.8:20.8.1–20.8.27.

Author Information

  1. 1

    Department of Biomolecular Mechanisms Max Planck Institute for Medical Research, Heidelberg, Germany

  2. 2

    Institute for Molecular Biophysics University of Mainz, Mainz, Germany

Publication History

  1. Published Online: 1 MAR 2005
  2. Published Print: FEB 2005


This unit provides guidelines on how to use steady-state fluorescence spectroscopy for the quantification of protein-protein interactions. The fluorescence of a protein is characterized by its excitation and emission spectra, quantum yield, and anisotropy. These parameters can change upon interaction with another protein and can be used to measure the extent of complex formation. The source of fluorescence can be an intrinsic fluorophore, such as tryptophan or tyrosine; a covalently attached fluorescent dye; or a fluorescent binding partner, such as a nucleotide or cofactor, that interacts specifically with the complex. Protocols are provided in this unit for determining affinity constants and stoichiometry values for protein-protein interactions using equilibrium titration experiments. In addition, fluorescent labeling of proteins is discussed, and an introduction to data analysis is provided. Most of the topics addressed in this unit can easily be applied to other spectroscopic methods or to the analysis of protein-ligand interactions.