Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells   

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Original research article

A brief version of this protocol appeared in:
18-Oct 2016


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: HaloTag, Live-cell single-molecule imaging, Polycomb, Cbx, Epigenetics, Janelia FluorTM dye


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).

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:
  1. Duc, H. N. and Ren, X. (2017). Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells. Bio-protocol 7(17): e2526. DOI: 10.21769/BioProtoc.2526.
  2. Zhen, C. Y., Tatavosian, R., Huynh, T. N., Duc, H. N., Das, R., Kokotovic, M., Grimm, J. B., Lavis, L. D., Lee, J., Mejia, F. J., Li, Y., Yao, T. and Ren, X. (2016). Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin. Elife 5.

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