Labelling HaloTag Fusion Proteins with HaloTag Ligand in Living Cells

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).

Specific labelling of biomolecules with fluorophores is the key for fluorescence singlemolecule 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 Fluor ™ dyes, such as Janelia Fluor ™ 549 (JF 549 ), are bright and photostable fluorescent HaloTag ligands (Grimm et al., 2015). This protocol describes how to label HaloTag-Cbx proteins with JF 549 for livecell single-molecule imaging, which was developed in the recent publication (Zhen et al.,

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35 mm glass bottom dish made in the laboratory (see Video 1 for making glassbottom dishes)

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Cell lines used in protocol: mouse embryonic stem cells and HEK293T cells

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Leukemia inhibitor factor (LIF, made in the laboratory)

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Following by overnight culture, the final confluency of the cells before was between 80-90%. Several concentrations (5 nM, 15 nM, and 30 nM) of JF 549 are used to incubate with cells for 15 min at 37 °C in 5% CO 2 (Notes 2 and 3) (see Video 3 for adding the JF 549 dye).

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Gently wash cells with ES medium (see Recipes) once and incubate in ES medium for 30 min at 37 °C and 5% CO 2 (see Video 4 for washing cells).

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Replace ES medium with live-cell imaging medium (see Recipes) (see Video 5 for adding live-cell imaging medium).

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Maintain 37 °C conditions during imaging by using a heater controller. Each plate should be imaged for the maximum of 1.5 h after placing on the microscope (see Video 6 for placing dishes on objective).

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The number of individual fluorescent spot per nucleus should be between 10-50 spots, controlled by adjusting the JF 549 dye concentrations (Notes 4 and 5) (see Video 7 for single-molecule imaging).

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Movies are then uploaded to u-track 2.0. Each cell is cropped from the larger movie. Cropped movies are processed (Note 6).

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Our data were analyzed using MATLAB with u-track 2.0 plug-in, detail guide can be found at http://www.utsouthwestern.edu/labs/danuser/software/ (the software and pdf file guide are included in the download).

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Representative images and movies can be found in Zhen et al., 2016.