An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli
Summary: Protein-protein interactions play important roles in regulating cellular functions. We present an optimized disulfide cross-linking protocol for testing predicted interactions of soluble or membrane proteins. Coexpression in E. coli of proteins with a single cysteine residue results in disu...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-03-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722008425 |
| _version_ | 1797977724891955200 |
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| author | Sandra Olenic Lee Kroos |
| author_facet | Sandra Olenic Lee Kroos |
| author_sort | Sandra Olenic |
| collection | DOAJ |
| description | Summary: Protein-protein interactions play important roles in regulating cellular functions. We present an optimized disulfide cross-linking protocol for testing predicted interactions of soluble or membrane proteins. Coexpression in E. coli of proteins with a single cysteine residue results in disulfide bond formation upon treating the cells with oxidants if the two proteins interact and the cysteine residues are near each other. Quantification of cross-linked proteins after immunoblot sensitively and reproducibly measures the interaction.For complete details on the use and execution of this protocol, please refer to Olenic et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-11T05:11:32Z |
| format | Article |
| id | doaj.art-1b2bbb2343e74793bb1608777ef23ddf |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-11T05:11:32Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-1b2bbb2343e74793bb1608777ef23ddf2022-12-25T04:19:18ZengElsevierSTAR Protocols2666-16672023-03-0141101962An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coliSandra Olenic0Lee Kroos1Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA; Corresponding authorDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA; Corresponding authorSummary: Protein-protein interactions play important roles in regulating cellular functions. We present an optimized disulfide cross-linking protocol for testing predicted interactions of soluble or membrane proteins. Coexpression in E. coli of proteins with a single cysteine residue results in disulfide bond formation upon treating the cells with oxidants if the two proteins interact and the cysteine residues are near each other. Quantification of cross-linked proteins after immunoblot sensitively and reproducibly measures the interaction.For complete details on the use and execution of this protocol, please refer to Olenic et al. (2022).1 : 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/S2666166722008425Molecular BiologyProtein BiochemistryProtein expression and purification |
| spellingShingle | Sandra Olenic Lee Kroos An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli STAR Protocols Molecular Biology Protein Biochemistry Protein expression and purification |
| title | An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli |
| title_full | An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli |
| title_fullStr | An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli |
| title_full_unstemmed | An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli |
| title_short | An optimized disulfide cross-linking protocol to determine interactions of proteins produced in Escherichia coli |
| title_sort | optimized disulfide cross linking protocol to determine interactions of proteins produced in escherichia coli |
| topic | Molecular Biology Protein Biochemistry Protein expression and purification |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722008425 |
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