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Updated: Aug 30, 2025
Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
Published on: September 7, 2011
Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry
Chingakham Ranjit Singh1, Rahul Jaiswal2, Carlos R Escalante2
1Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA; Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Abstract:
eIF5-mimic protein (5MP) controls translation through its interaction with eukaryotic translation initiation factor (eIF) 2 and eIF3 and alters non-AUG translation rates for oncogenes in cancer and repeat expansions in neurodegenerative disease. To precisely evaluate the effect of 5MP mutations on binding affinity against eIFs, here we describe two label-free protocols of affinity measurement for 5MP binding to eIF2 or eIF3 protein segments, termed isothermal titration calorimetry (ITC) and bio-layer interferometry (BLI), starting with how to purify proteins used. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).
