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SH2 Domains pp 291-305 | Cite as

Calorimetric Measurement of SH2 Domain Ligand Affinities

  • Marissa A. McKercher
  • Deborah S. WuttkeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1555)

Abstract

Isothermal titration calorimetry (ITC) has emerged as a leading approach in the characterization of protein/ligand interactions. This technique measures the heat change of a system upon binding of a ligand to a biomolecule, and thereby requires no immobilization, intrinsic fluorescence, or labeling of any kind of either species. If properly designed, a single experiment can not only measure the binding affinity, but also determine additional binding and thermodynamic parameters, including the enthalpy, entropy, and the stoichiometry of the interaction. Here, we describe the protocol for the collection of calorimetric data for the binding of peptides to SH2 protein domains.

Key words

Isothermal titration calorimetry ITC SH2 domain Peptide Binding affinity Thermodynamics Enthalpy Entropy 

Notes

Acknowledgments

We would like to thank Dr. Annette Erbse for her generous feedback and technical support. We would also like to thank our collaborators Dr. Zhongping Tan and Dr. Xiaoyang Guan for the phosphopeptide synthesis, as well as Dr. Julie Forman-Kay for kindly providing us with a PLCγ1-pET11d construct. This work was supported by NSF grant MCB1121842 (to D.S.W.).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of ColoradoBoulderUSA

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