Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein
Summary: 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 mutat...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-09-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722004956 |
| _version_ | 1818486812316794880 |
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| author | Chingakham Ranjit Singh Rahul Jaiswal Carlos R. Escalante Katsura Asano |
| author_facet | Chingakham Ranjit Singh Rahul Jaiswal Carlos R. Escalante Katsura Asano |
| author_sort | Chingakham Ranjit Singh |
| collection | DOAJ |
| description | Summary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-12-10T16:28:00Z |
| format | Article |
| id | doaj.art-758261de9fe24f2490d42b41fc075812 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-12-10T16:28:00Z |
| publishDate | 2022-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-758261de9fe24f2490d42b41fc0758122022-12-22T01:41:37ZengElsevierSTAR Protocols2666-16672022-09-0133101615Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic proteinChingakham Ranjit Singh0Rahul Jaiswal1Carlos R. Escalante2Katsura Asano3Molecular 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; Corresponding authorDepartment of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USADepartment of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA; Corresponding authorMolecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA; Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan; Hiroshima Research Center for Healthy Aging, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan; Corresponding authorSummary: 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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722004956BiophysicsMolecular biology |
| spellingShingle | Chingakham Ranjit Singh Rahul Jaiswal Carlos R. Escalante Katsura Asano Label-free protocol to quantify protein affinity using isothermal titration calorimetry and bio-layer interferometry of a human eIF5-mimic protein STAR Protocols Biophysics Molecular biology |
| 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 | Biophysics Molecular biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722004956 |
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