Published: Vol 7, Iss 17, Sep 5, 2017 DOI: 10.21769/BioProtoc.2526 Views: 11347
Reviewed by: Arsalan DaudiAnonymous reviewer(s)
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Abstract
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
Keywords: HaloTagBackground
Molecular processes of living organisms are intrinsically dynamics. Direct observation of the molecular processes in living cells is critical for quantitatively understanding of how biological systems function. Recent advances in fluorescence microscopy and fluorescent labelling enable to visualize trajectories of individually single molecules in living cells, providing insights about dynamic interactions and assemblies of biological molecules (Kusumi et al., 2014; Liu et al., 2015; Tatavosian et al., 2015; Cuvier and Fierz, 2017). Specific labelling of biomolecules with fluorophores is the key for fluorescence single-molecule imaging. HaloTag is self-labeling tag proteins that can be coupled to synthetic dyes in living cells (Los et al., 2008). The reaction occurs rapidly in living cells and the formed covalent bond is specific and irreversible. This technique has been utilized to study the genetic information flow in vivo, and to measure the kinetic of gene regulation in living mammalian cells (Liu et al., 2015; Zheng and Lavis, 2017). Janelia FluorTM dyes, such as Janelia FluorTM 549 (JF549), are bright and photostable fluorescent HaloTag ligands (Grimm et al., 2015). This protocol describes how to label HaloTag-Cbx proteins with JF549 for live-cell single-molecule imaging, which was developed in the recent publication (Zhen et al., 2016).
Materials and Reagents
Equipment
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Procedure
Data analysis
Notes
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Acknowledgments
This work was supported, in whole or in part, by the National Cancer Institute of the National Institutes of Health under Award Number R03CA191443 (to XR). This work was also supported by grants from the CU-Denver Office Research Service (to XR) and the American Cancer Society Grant IRG 57-001-53 subaward (to XR). This protocol was originally developed in Zhen et al., 2016.
References
Article Information
Copyright
Duc and Ren . This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Developmental Biology > Cell signaling
Biochemistry > Protein > Labeling
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