Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein

STAR Protoc. 2022 Aug 16;3(3):101615. doi: 10.1016/j.xpro.2022.101615. eCollection 2022 Sep 16.

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).

Keywords: Biophysics; Molecular biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Calorimetry
  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factor-3
  • Eukaryotic Initiation Factor-5*
  • Humans
  • Interferometry
  • Neurodegenerative Diseases*

Substances

  • Eukaryotic Initiation Factor-2
  • Eukaryotic Initiation Factor-3
  • Eukaryotic Initiation Factor-5