Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates
Summary: Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for t...
| Main Authors: | , |
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| Format: | Article |
| Language: | English |
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Elsevier
2023-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166723006834 |
| _version_ | 1827761736851128320 |
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| author | Nathalie Djaja Sua Myong |
| author_facet | Nathalie Djaja Sua Myong |
| author_sort | Nathalie Djaja |
| collection | DOAJ |
| description | Summary: Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions.For complete details on the use and execution of this protocol, please refer to Djaja et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-03-11T10:20:08Z |
| format | Article |
| id | doaj.art-e6f1043e3b9b42b787cee3418fbb3775 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-03-11T10:20:08Z |
| publishDate | 2023-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-e6f1043e3b9b42b787cee3418fbb37752023-11-16T06:10:57ZengElsevierSTAR Protocols2666-16672023-12-0144102716Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysatesNathalie Djaja0Sua Myong1Program in Cell, Molecular, Developmental Biology, and Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA; Department of Biology, Johns Hopkins University, Baltimore, MD 21218; Corresponding authorDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218; Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218; Corresponding authorSummary: Mutations in intrinsically disordered proteins drive the irreversible formation of pathological aggregates, a hallmark of neurodegenerative diseases. Here, we present a protocol to pull down fluorescently tagged proteins to characterize their basal oligomeric states. We describe steps for transfection and cell lysis, single-molecule slide preparation and pull-down, and oligomer dissolution. This protocol enables visualization of protein oligomers with single-molecule resolution. In addition, differences in oligomerization may provide insight on condensation or aggregation propensity in differing mutated or cell stress conditions.For complete details on the use and execution of this protocol, please refer to Djaja et al.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/S2666166723006834Single-molecule AssaysCell-based AssaysMolecular Biology |
| spellingShingle | Nathalie Djaja Sua Myong Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates STAR Protocols Single-molecule Assays Cell-based Assays Molecular Biology |
| title | Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates |
| title_full | Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates |
| title_fullStr | Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates |
| title_full_unstemmed | Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates |
| title_short | Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates |
| title_sort | protocol for single molecule pull down of fluorescently tagged oligomers from cell lysates |
| topic | Single-molecule Assays Cell-based Assays Molecular Biology |
| url | http://www.sciencedirect.com/science/article/pii/S2666166723006834 |
| work_keys_str_mv | AT nathaliedjaja protocolforsinglemoleculepulldownoffluorescentlytaggedoligomersfromcelllysates AT suamyong protocolforsinglemoleculepulldownoffluorescentlytaggedoligomersfromcelllysates |