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

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 b...

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Autores principales: Singh, Chingakham Ranjit, Jaiswal, Rahul, Escalante, Carlos R., Asano, Katsura
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403556/
https://www.ncbi.nlm.nih.gov/pubmed/36035794
http://dx.doi.org/10.1016/j.xpro.2022.101615
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author Singh, Chingakham Ranjit
Jaiswal, Rahul
Escalante, Carlos R.
Asano, Katsura
author_facet Singh, Chingakham Ranjit
Jaiswal, Rahul
Escalante, Carlos R.
Asano, Katsura
author_sort Singh, Chingakham Ranjit
collection PubMed
description 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).
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spelling pubmed-94035562022-08-26 Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein Singh, Chingakham Ranjit Jaiswal, Rahul Escalante, Carlos R. Asano, Katsura STAR Protoc Protocol 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). Elsevier 2022-08-16 /pmc/articles/PMC9403556/ /pubmed/36035794 http://dx.doi.org/10.1016/j.xpro.2022.101615 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Singh, Chingakham Ranjit
Jaiswal, Rahul
Escalante, Carlos R.
Asano, Katsura
Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title_full Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title_fullStr Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title_full_unstemmed Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title_short Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
title_sort label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eif5-mimic protein
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9403556/
https://www.ncbi.nlm.nih.gov/pubmed/36035794
http://dx.doi.org/10.1016/j.xpro.2022.101615
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