Remenyi, R, Li, R orcid.org/0000-0002-8733-1624 and Harris, M orcid.org/0000-0002-9821-1003 (2019) On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates. Bio-protocol, 9 (4). ARTN e3177. ISSN 2331-8325
Abstract
Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. In contrast, self-labeling enzyme tags such as the commercial SNAP-tag, a modified version of human O6-alkylguanine-DNA-alkyltransferase, covalently link synthetic ligands, which users can add on demand. The first two protocols presented here build on previously published protocols for fluorescent labeling in pulse-chase and quench-pulse-chase experiments; the combination of fluorescent labeling with advanced light microscopy visualizes the dynamic turnover of the SNAP-tagged viral protein in intact mammalian cells. A third protocol also outlines how to inspect cellular lysates microscopically for detergent-resistant assemblies of the labeled viral protein. These protocols showcase the flexibility of the SNAP-based labeling system for tracking a viral protein-of-interest in live cells, intact fixed cells, and cell lysates. Moreover, the protocols employ recently developed commercial microscopes (e.g., Airyscan microscopy) that balance resolution, speed, phototoxicity, photobleaching, and ease-of-use.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Keywords: | Fusion proteins; Stress granules; RNPs; Site-specific protein labeling; Chemical labeling; Cell biology; Bioimaging; Live cell imaging; Chikungunya; CHIKV; Alphavirus; Arbovirus; nsP3 |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Virology 1 (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 096670/Z/11/Z |
Depositing User: | Symplectic Publications |
Date Deposited: | 14 Mar 2019 13:20 |
Last Modified: | 14 Mar 2019 13:21 |
Status: | Published |
Publisher: | Bio-Protocol |
Identification Number: | 10.21769/BioProtoc.3177 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:143627 |