A stock solution of HaloTag TMR Ligand (Promega, Madison, WI) was diluted in supplemented DMEM to a final concentration of 5 nM. After incubation posttransfection, all medium was removed from the cells and replaced with supplemented DMEM containing 5 nM HaloTag TMR Ligand (Promega) and 5 μM SNAP-Cell 505-Star (New England Biolabs, Ipswich, MA) before incubation for 30 min at 37°C. Cells were then washed with 3 × 1 mL medium and incubated for an additional 30 min in 1 mL of medium before a final wash with 3 × 1 mL medium.
Halotag tmr ligand
Manufactured by Promega
Sourced in United States
The HaloTag TMR ligand is a fluorescent dye that can be used to label proteins that have been genetically modified to include a HaloTag protein fusion. The ligand binds covalently to the HaloTag protein, allowing visualization and detection of the tagged protein.
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Lab products found in correlation
Halotag antibody, by Promega (4 mentions)
Snap cell tmr star, by New England Biolabs (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Typhoon trio imager, by GE Healthcare (2 mentions)
Fusion alpha fp ht, by PerkinElmer (2 mentions)
Facs aria 3 system, by BD (2 mentions)
Janelia fluor 549, by Promega (2 mentions)
Alexa 488 conjugated anti rat antibody, by Thermo Fisher Scientific (2 mentions)
Axio observer d1 microscope, by Zeiss (2 mentions)
Alexa fluor 488 labeled goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568 labeled goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alpha plan apochromat 100 1.40 oil immersion objective, by Zeiss (2 mentions)
Brightline single band laser filter set, by IDEX Corporation (2 mentions)
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
67 protocols using halotag tmr ligand
1
Dual Fluorescent Protein Labeling
2
Halo-tag Protein Labeling in D.mel-2 Cells
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Protein extracts of D.mel-2 cells expressing Halo-tag fusions were prepared as previously described (Weidmann and Goldstrohm, 2012 (link)). Extracts were then incubated with 100 nM Halo-tag TMR Ligand (Promega) for 30 min on ice, protected from light. After labeling, extracts were separated via SDS polyacrylamide gel electrophoresis and labeled proteins were imaged with a Typhoon Trio imager (GE Healthcare).
3
Visualization of Halo-tagged Myo19 in U2OS cells
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Transfections were performed using Polyethylenimine (PEI) purchased from Weis Scientific – PolySciences (Warrington, PA, USA). Adherent U2OS cells were plated a day before transfection on glass bottom dishes purchased from In-vitro Scientific (Mountain View, CA, USA) and allowed to adhere overnight. Plasmid DNA and PEI were diluted separately in 150 mM NaCl, combined and complex formation was allowed for 25 min at RT before addition to the cells and incubation overnight. Hoechst 33342 (0.75 µg/ml) was added 15 min prior to imaging. To visualize Halo tagged Myo19, HaloTag TMR ligand (Promega) was added to cells transfected with Halo tagged Myo19 at a final concentration of 50 nM and incubated overnight. Images were taken using LSM710 at ×40 magnification or INCELL2000 at ×60 for live cells in an environmental chamber or using LSM710 at ×63 for fixed samples.
4
Immunofluorescence Assay for HBV Proteins
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Immunofluorescence assay was basically performed as described previously [27 (link)]. Primary antibodies used in the study included rabbit anti-HBc (Neomarkers, RB-1413-A), anti-HDAg, anti-NTCP (Sigma, HPA042727), mouse anti-c-Myc (Santa Cruz, sc-40), and anti-α-tubulin (Sigma, T5168). Alexa Flour555-, Alexa Flour488-, or Alexa Flour647-conjugated secondary antibodies (Invitrogen) were utilized together with DAPI to visualize the nucleus. For Halo tag, live cells were treated with cell-permeant Halotag TMR ligand (Promega, G8251) before paraformaldehyde fixation. Microscopic examination of the infected cells or preS1 binding was performed by fluorescence microscopy (KEYENCE, BZ-X710); the observation of the subcellular localization was performed using a high-resolution confocal microscope (Leica, TCS 159 SP8) as described previously [27 (link)].
5
Halotag Fusion Protein Labeling
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Protein extracts from HEK293 cells expressing Halotag fusions were harvested from each well of a 96-well plate in 20 µL of lysis buffer (0.5% Igepal CA-630 [USB], 50 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 2 mM MgCl2, 150 mM NaCl) with 1× Protease Inhibitor cocktail (Promega) and mixed with 900 nM Halotag TMR Ligand (Promega) for 30 min on ice, protected from light. For labeling of FLAG IPs, refer to FLAG IP methods. After labeling, extracts were separated via SDS-polyacrylamide (12%) gel electrophoresis and detected by fluorescence imaging with a Typhoon Trio imager (GE Healthcare).
6
Monitoring Autophagy Flux Using HaloTag-LC3
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HaloTag-LC3 assays were performed as previously described (Takahashi et al., 2018 (link)). Briefly, cells were transfected with HaloTag-LC3 for 24 h and treated with 20 μM digitonin in KHM buffer at 37°C for 2 min. Then cells were incubated with membrane-impermeable ligand (MIL) HaloTag® Alexa Fluor® 488 ligand (G1001, Promega) at 37°C for 15 min. Cells were fixed in 4% PFA for 20 min at room temperature, washed 3 times with PBS, and stained with membrane-permeable ligand (MPL) HaloTag® TMR ligand (G8251, Promega) for 30 min at room temperature. Coverslips were mounted on microscope slides with DAPI in 50% glycerol and examined by a confocal microscope (LSM 880 Meta plus Zeiss Axiovert zoom, Zeiss).
7
In Vivo Cross-linking and Covalent Capture
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Cross-linking was performed as described in (Soh et al., 2019 (link)). Cultures were grown in SMG to OD600 of 0.03–0.04 at 37°C. Cells were mixed with ice and harvested by centrifugation. Sample handling and preparation were performed on ice and cold at every step. Cells were washed in PBSG (PBS with 0.1% [v/v] glycerol). Samples were resuspended in 1 mL PBSG. 1.25 OD600 equivalents were taken and pelleted by centrifugation. Pellets were resuspended in 30 μL PBSG. Cross-linking agent (SMCC (Thermo) or BMOE (Thermo) was added to a final concentration of 0.5 mM and mixed by vortexing. Reactions were incubated on ice for 10 minutes and then quenched by the addition of 0.5 mM final concentration 2-mercaptoethanol with subsequent incubation for 2 minutes. Samples were supplemented with additives at the indicated final concentrations: benzonase (750 U/mL; Sigma), 5 μM HaloTag-TMR ligand (Promega), Ready-Lyse Lysozyme (47 U/μL; Epicentre), and 1× PIC (Sigma-Aldrich). Samples were incubated at 37°C for 30 minutes under light protection. Samples were supplemented with LPS loading dye and denatured at 70°C for 5 minutes. Samples were run on 3-8% Tris-acetate gels (Invitrogen) at a constant power output of 35 mA at 4°C. In-gel fluorescence was imaged using an Amersham Typhoon (GE Healthcare) with a Cy3 DIGE filter. Quantification was performed using ImageQuant (GE Healthcare).
8
Single-molecule Imaging of Lyn-Halotag
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HeLa cells grown on glass coverslips (Matsunami) in a 6-well plate were transfected with Lyn11-Halotag using Lipofectamine 2000 (Invitrogen). Afters 4 h, the culture medium was replaced with DMEM and the cells were incubated at 37 °C for 24 h. The culture medium was exchanged with Opti-MEM (Gibco), and the cells were incubated at 37 °C. After 2 h, the cells were washed once with Opti-MEM and incubated with 0.03 nM of Halotag TMR ligand (Promega) in Opti-MEM for 30 min in a CO2 incubator. The cells were then washed three times with Opti-MEM and single-molecule imaging was performed using a TIRF microscope. Single particle detection and estimation of diffusion constants were done using ICY and PNN algorithm, respectively.
9
Visualizing Protease and Tubulin in Cells
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To visualize the protease within the HaloTag-HTRA1 and HaloTag-S328A overexpressing cells we performed a rapid labelling protocol according to the manufacturer's instructions (Promega). Briefly, cells were incubated with the Halotag TMR ligand (G8251, Promega) at a concentration 5 μM during 30 min at 37 °C, washed twice with fresh media, incubated again for 30 min at 37 °C with fresh media and imaged using phenol red free fresh MEM (41,061, Gibco).
To stain for tubulin in living cells, samples were incubated for 6 h at 37 °C with SiR-Tubulin at a concentration of 200 nM (SC006, Spirichrome). Cells were then washed twice with fresh media and incubated with the Halotag ligand following the protocol previously described. Prior to imaging, cells were stained with Hoechst following the manufacturer's specifications (33,342, Lifetechnologies).
To stain for tubulin in living cells, samples were incubated for 6 h at 37 °C with SiR-Tubulin at a concentration of 200 nM (SC006, Spirichrome). Cells were then washed twice with fresh media and incubated with the Halotag ligand following the protocol previously described. Prior to imaging, cells were stained with Hoechst following the manufacturer's specifications (33,342, Lifetechnologies).
10
Kinetic Analysis of GFP-ssrA Degradation by ClpA and ClpP
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