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HaloTag

HaloTag: An innovative protein tag technology that enables precise labeling, visualization, and manipulation of target proteins in live cells.
Leveraging artificial intelligence, PubCompare.ai streamlines HaloTag experiments, helping researchers locate the best protocols from literature, preprints, and patents through intelligent comparisons.
This ensures reproducible and accurate HaloTag-based research, unlocking the full power of this versatile technology and achieving maximum experimental results.

Most cited protocols related to «HaloTag»

1
Fluorescent Labeling of Live Cells
Cited 87 times
HeLa cells (ATCC) and U2OS cells (ATCC) were cultured in Dulbecco’s modified eagle medium (DMEM; Life Technologies) supplemented with 10% v/v fetal bovine serum (FBS; Life Technologies), 1 mM GlutaMax (Life Technologies), and 1 mM sodium pyruvate (Sigma) and maintained at 37 °C in a humidified 5% v/v CO2 environment. These cell lines undergo regular mycoplasma testing by the Janelia Cell Culture Facility. Cells were transfected with HaloTag–H2B, HaloTag–tubulin, SnapTag–TetR, or SnapTag–H2B using an Amaxa Nucleofector (Lonza). Before the imaging experiments, transfected cells were transferred onto a No.1 coverslip (Warner Instruments) that was cleaned by Piranha solution (3:1 v/v mixture of concentrated H2SO4 and 30% v/v hydrogen peroxide). To label live cells with the HaloTag or SnapTag ligands, compounds 9, 10, 27, 28, 29, 30, or 31 were added to the growth medium and the samples incubated for 15 min. Labeling concentrations were typically 100–500 nM for confocal, wide-field, and dSTORM experiments and 5–50 nM for single-molecule tracking experiments. Cells were then washed briefly with PBS (1×) and then incubated in DMEM–FBS for an additional 15 min. Before imaging, the cells were washed briefly with PBS (3×) and placed in fresh DMEM–FBS for imaging. All washes were omitted in the “no wash” experiments. For nuclear staining, cells were incubated in PBS for 5 min (2×), and then incubated in PBS containing 5 μM DRAQ5 (Cell Signaling) for 5 min, followed by brief wash with PBS (1×). During all imaging experiments, cells were maintained at 37 °C in a humidified 5% CO2 v/v environment supplied by a live-cell incubator (TOKAI HIT).
Grimm J.B., English B.P., Chen J., Slaughter J.P., Zhang Z., Revyakin A., Patel R., Macklin J.J., Normanno D., Singer R.H., Lionnet T, & Lavis L.D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature methods, 12(3), 244-250.
Publication 2014
Cell Culture Techniques Cell Lines Cells Culture Media Eagle HaloTag HeLa Cells Ligands Mycoplasma Peroxide, Hydrogen Piranhas Pyruvate Sodium Tubulin
2
Fluorescent Labeling of Live Cells
Cited 82 times
HeLa cells (ATCC) and U2OS cells (ATCC) were cultured in Dulbecco’s modified eagle medium (DMEM; Life Technologies) supplemented with 10% v/v fetal bovine serum (FBS; Life Technologies), 1 mM GlutaMax (Life Technologies), and 1 mM sodium pyruvate (Sigma) and maintained at 37 °C in a humidified 5% v/v CO2 environment. These cell lines undergo regular mycoplasma testing by the Janelia Cell Culture Facility. Cells were transfected with HaloTag–H2B, HaloTag–tubulin, SnapTag–TetR, or SnapTag–H2B using an Amaxa Nucleofector (Lonza). Before the imaging experiments, transfected cells were transferred onto a No.1 coverslip (Warner Instruments) that was cleaned by Piranha solution (3:1 v/v mixture of concentrated H2SO4 and 30% v/v hydrogen peroxide). To label live cells with the HaloTag or SnapTag ligands, compounds 9, 10, 27, 28, 29, 30, or 31 were added to the growth medium and the samples incubated for 15 min. Labeling concentrations were typically 100–500 nM for confocal, wide-field, and dSTORM experiments and 5–50 nM for single-molecule tracking experiments. Cells were then washed briefly with PBS (1×) and then incubated in DMEM–FBS for an additional 15 min. Before imaging, the cells were washed briefly with PBS (3×) and placed in fresh DMEM–FBS for imaging. All washes were omitted in the “no wash” experiments. For nuclear staining, cells were incubated in PBS for 5 min (2×), and then incubated in PBS containing 5 μM DRAQ5 (Cell Signaling) for 5 min, followed by brief wash with PBS (1×). During all imaging experiments, cells were maintained at 37 °C in a humidified 5% CO2 v/v environment supplied by a live-cell incubator (TOKAI HIT).
Grimm J.B., English B.P., Chen J., Slaughter J.P., Zhang Z., Revyakin A., Patel R., Macklin J.J., Normanno D., Singer R.H., Lionnet T, & Lavis L.D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature methods, 12(3), 244-250.
Publication 2014
Cell Culture Techniques Cell Lines Cells Culture Media Eagle HaloTag HeLa Cells Ligands Mycoplasma Peroxide, Hydrogen Piranhas Pyruvate Sodium Tubulin
3
Coelenterazine-based Luminescence Screening
Cited 53 times
Random libraries were generated
by error-prone PCR (average of 2–3 mutations per clone). Library
1 (phase 1; template = Oluc-N166R) was screened (4,400 variants) with
coelenterazine. Library 2 (phase 2; template = C1A4E) was screened
(4,400 variants) with 11 novel coelenterazine analogues: 3840, 3841,
3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900 (Supplementary Figure s4). The 11 analogues represented
substitutions at positions 2, 6, and 8 and were considered to be representative
of the entire set of 24 compounds; 2,200 variants were screened with
compounds 3896 and 3894 (Supplementary Figure
s4). All hits (improved luminescence) were screened again with
the remaining coelenterazine analogues. Library 3 (phase 3; template
= C1A4E + Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the
context of a mouse Id-X-HaloTag (where X = library) using coelenterazine
and furimazine (Figure 1c). Library screens
were performed on a Freedom robotic workstation (Tecan) as follows:
induced bacterial cultures (in 96-well microtiter plates) were lysed
with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X
Passive Lysis Buffer (PLB, Promega), 0.3 mg mL–1 lysozyme, and 0.002 units of RQ1 DNase (Promega). Assay reagent
containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol,
and 20 μM substrate was then added to equal volumes of lysate.
Samples were measured on a GENios Pro luminometer (Tecan). Secondary
screening to confirm hits (defined as those variants producing greater
luminescence compared to that of the parental clone) and to test combination
sequences was completed using a similar protocol but in manual fashion
and in triplicate.
Hall M.P., Unch J., Binkowski B.F., Valley M.P., Butler B.L., Wood M.G., Otto P., Zimmerman K., Vidugiris G., Machleidt T., Robers M.B., Benink H.A., Eggers C.T., Slater M.R., Meisenheimer P.L., Klaubert D.H., Fan F., Encell L.P, & Wood K.V. (2012). Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate. ACS Chemical Biology, 7(11), 1848-1857.
Publication 2012
Bacteria Biological Assay Buffers cDNA Library CDTA Clone Cells coelenterazine Deoxyribonucleases furimazine HaloTag hen egg lysozyme HEPES Luminescence Mice, House Mutation N-dodecyl-L-lysine amide Parent Promega Tergitol Thiourea
4
HaloTag Ligand Absorbance Measurement
Cited 18 times
Absorbance measurements were performed in 1 mL quartz cuvettes. HaloTag protein was used as a 100 μM solution in 75 mM NaCl, 50 mM TRIS·HCl, pH 7.4 with 50% v/v glycerol (TBS–glycerol). HaloTag ligands 27 and 28 (5 μM) were dissolved in 10 mM HEPES, pH 7.3 containing 0.1 mg·mL−1 CHAPS. An aliquot of HaloTag protein (1.5 equiv) or an equivalent volume of TBS–glycerol blank was added and the resulting mixture was incubated until consistent absorbance signal was observed (~30 min). Additional HaloTag protein did not elicit an increase in absorbance (not shown). Absorbance scans are averages (n = 2).
Grimm J.B., English B.P., Chen J., Slaughter J.P., Zhang Z., Revyakin A., Patel R., Macklin J.J., Normanno D., Singer R.H., Lionnet T, & Lavis L.D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature methods, 12(3), 244-250.
Publication 2014
3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate Glycerin HaloTag HEPES Ligands Proteins Quartz Radionuclide Imaging Sodium Chloride Synapsin I Tromethamine
5
HaloTag Ligand Absorbance Measurement
Cited 18 times
Absorbance measurements were performed in 1 mL quartz cuvettes. HaloTag protein was used as a 100 μM solution in 75 mM NaCl, 50 mM TRIS·HCl, pH 7.4 with 50% v/v glycerol (TBS–glycerol). HaloTag ligands 27 and 28 (5 μM) were dissolved in 10 mM HEPES, pH 7.3 containing 0.1 mg·mL−1 CHAPS. An aliquot of HaloTag protein (1.5 equiv) or an equivalent volume of TBS–glycerol blank was added and the resulting mixture was incubated until consistent absorbance signal was observed (~30 min). Additional HaloTag protein did not elicit an increase in absorbance (not shown). Absorbance scans are averages (n = 2).
Grimm J.B., English B.P., Chen J., Slaughter J.P., Zhang Z., Revyakin A., Patel R., Macklin J.J., Normanno D., Singer R.H., Lionnet T, & Lavis L.D. (2015). A general method to improve fluorophores for live-cell and single-molecule microscopy. Nature methods, 12(3), 244-250.
Publication 2014
3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate Glycerin HaloTag HEPES Ligands Proteins Quartz Radionuclide Imaging Sodium Chloride Synapsin I Tromethamine

Most recents protocols related to «HaloTag»

1
Synthesis and Characterization of Halo-SiR Fluorescent Probe

Protocol full text hidden due to copyright restrictions

Open the protocol to access the free full text link

Publication 2023
1H NMR Amines Chromatography Gel Chromatography HaloTag Mass Spectrometry Pressure Silica Gel Silicon Dioxide Solvents Spectrometry
2
Vesicle Tracking and Surface Analysis
Vesicle tracking and surface analysis were analyzed by Imaris 9.2.1 software package (Bitplane). Surface analysis was done to determine the amounts of effector-positive vesicles inside an infected cell. By the surface tool, data acquired by LCI were analyzed in the red (PipB2-HaloTag-TMR) channel. Vesicle volume was adjusted using auto-threshold and smoothing of 0.1, and SIF volume was adjusted using auto-threshold and smoothing of 0.3. For vesicle tracking, the spot tool was used. Spot detection was performed with the following parameters XY diameter: 0.75 µm, active model PSF elongation: 1.5 µm, and background subtraction. For tracking the autoregressive motion algorithm was chosen with a maximum gap size of 3.
Göser V., Sander N., Schulte M., Scharte F., Franzkoch R., Liss V., Psathaki O.E, & Hensel M. (2023). Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nature Communications, 14, 1240.
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Publication 2023
Cells HaloTag
3
Quantifying Intravacuolar Salmonella Trafficking
The FIJI package51 (link) was used to determine intensity profiles of accumulated SifA-HaloTag trajectories on SIF. Using the line tool and subsequently, the plot profile tool, the intensity profile of the trajectories labeling the SIF was calculated and the relative distances in pixel were compared.
Göser V., Sander N., Schulte M., Scharte F., Franzkoch R., Liss V., Psathaki O.E, & Hensel M. (2023). Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nature Communications, 14, 1240.
Full text: Click here
Publication 2023
HaloTag
4
Salmonella Typhimurium Effector Protein Analysis
Infection experiments were performed using Salmonella enterica serovar Typhimurium (STM) NCTC 12023 strain as WT and isogenic mutant strains (Table 1). Mutagenesis was carried out as described elsewhere45 (link). In short, strains were constructed using λ Red-mediated mutagenesis and the resistance cassette was removed using FLP-mediated recombination. Mutant strains deficient in effector genes harboring plasmids encoding the corresponding effector fused to HaloTag (Table 2) were used for microscopic analysis.

Salmonella enterica serovar Typhimurium strains used in this study

DesignationRelevant characteristicsReference
NCTC 12023Wild typeLab stock
MvP388ΔsscB sseF::FRT52 (link)
MvP392ΔsseJ::FRT53 (link)
MvP503ΔsifA::FRT54 (link)
MvP742ΔsteC::FRTThis study
MvP1900ΔsseJ::aph ΔsifA::FRT45 (link)
MvP1944ΔpipB2::FRT44 (link)
MvP1948ΔsseF::aph ΔsifA::FRTThis study
MvP1980ΔsseF::FRT55 (link)

Plasmids used in this study

DesignationRelevant genotypeSource/reference
p2095PsseA::sscB sseF::M4552 (link)
p2129PsseJsseJ::M4541 (link)
p2621PpipB2pipB2::M4518 (link)
p2643PsseA::sscB sseF::HA52 (link)
p2888PsseA::sscB sseF::HA sseG::M4552 (link)
p3805PCMV::LAMP1-miniSOG-mCherrythis study
p3806PCMV::LAMP1-tdminiSOG-mCherrythis study
p3991LAMP1::HaloTag::GFP9 (link)
p4305PsifAsifA::L16::HaloTag::HA16 (link)
p4118PsseAsscB sseF::L16::HaloTag::HA16 (link)
p4286PsseJsseJ::L16::HaloTag::HA16 (link)
p4295PpipB2pipB2::L16::HaloTag::HA16 (link)
p5059PsteCsteC::L16::HaloTag::HAThis study
p5065PsseA::sscB sseF::3HAThis study
p6172PpipB2pipB2 ΔCC::L16::HaloTag::HAthis study
p6173PsopD2sopD2 ΔCC::L16::HaloTag::HAthis study
p6187PsifAsifA Δ331-336::L16::HaloTag::HAthis study
p6203PsseJsseJ ΔCC::L16::HaloTag::HAthis study
Göser V., Sander N., Schulte M., Scharte F., Franzkoch R., Liss V., Psathaki O.E, & Hensel M. (2023). Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nature Communications, 14, 1240.
Full text: Click here
Publication 2023
Genes HaloTag Infection lysosomal-associated membrane protein 1, human Microscopy Mutagenesis Plasmids Recombination, Genetic Salmonella enterica Strains
5
Plasmid Construction and Mutagenesis
Plasmids were constructed as described previously16 (link) using oligonucleotides listed in Supplementary Table 1. Bacterial strains were cultured in Luria-Bertani broth (LB) containing 50 µg ml−1 Carbenicillin (Roth, 6344.3).
For generation of a plasmid encoding triple HA-tagged SseF, p2643 (sscB sseF::HA) was used and sseF::HA on p2643 was replaced by sseF::3HA using Gibson assembly GA. Primers for generation of vector fragment, check primers and sequence of synthetic sseF::3xHA (gBlocks, IDT) are listed in Supplementary Table 1.
Mutagenesis was performed for plasmids p4286, p4295, p4300, and p4305 encoding sseJ::HaloTag, pipB2::HaloTag, sopD2::HaloTag or sifA::HaloTag, respectively, using primers listed in Supplementary Table 1. Deletion of codons 331-336 in sifA removes the previously described prenylation site24 (link) and was expected to reduce membrane interaction. Mutations Y281D Y284D K288D (sopD2), N309D L312D V316D (sseJ), and Y54D L57D M61D (pipB2) affect coiled-coil (CC) domains that were reported to contribute to membrane interactions of translocated T3SS effector proteins25 (link). Site-directed mutagenesis was performed using the Q5 SDM kit (NEB, E0554S), and resulting plasmids were confirmed by sequencing (SeqLab, Göttingen).
Göser V., Sander N., Schulte M., Scharte F., Franzkoch R., Liss V., Psathaki O.E, & Hensel M. (2023). Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nature Communications, 14, 1240.
Full text: Click here
Publication 2023
Bacteria Carbenicillin Cloning Vectors Codon Deletion Mutation HaloTag Mutagenesis Mutagenesis, Site-Directed Mutation Oligonucleotide Primers Oligonucleotides Plasmids Prenylation Strains Tissue, Membrane

Top products related to «HaloTag»

1
Halotag tmr ligand 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.
2
Lipofectamine 2000 by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Canada, Japan, France, Italy, Switzerland, Australia, Spain, Belgium, Denmark, Singapore, India, Netherlands, Sweden, New Zealand, Portugal, Poland, Israel, Lithuania, Hong Kong, Argentina, Ireland, Austria, Czechia, Cameroon, Taiwan, Province of China, Morocco
Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
3
Magne halotag beads by Promega
Sourced in United States
Magne® HaloTag® Beads are magnetic affinity beads designed for the purification and detection of HaloTag®-fusion proteins. The beads are coated with a modified HaloTag® ligand, allowing for the efficient capture and immobilization of HaloTag®-tagged proteins.
4
Lipofectamine 3000 by Thermo Fisher Scientific
Sourced in United States, China, Germany, Japan, United Kingdom, France, Canada, Italy, Australia, Switzerland, Denmark, Spain, Singapore, Belgium, Lithuania, Israel, Sweden, Austria, Moldova, Republic of, Greece, Azerbaijan, Finland
Lipofectamine 3000 is a transfection reagent used for the efficient delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of mammalian cell types. It facilitates the entry of these molecules into the cells, enabling their expression or silencing.
5
Fugene hd transfection reagent by Promega
Sourced in United States, Germany, United Kingdom, Japan, China, France, Canada, Australia, Italy, Switzerland
FuGENE HD is a transfection reagent that facilitates the delivery of nucleic acids, such as plasmid DNA, into mammalian cells. It is designed to enhance transfection efficiency in a variety of cell lines.
6
Opti mem by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, China, Japan, Canada, France, Switzerland, Italy, Australia, Belgium, Spain, Denmark, Ireland, Netherlands, Holy See (Vatican City State), Israel
Opti-MEM is a cell culture medium designed to support the growth and maintenance of a variety of cell lines. It is a serum-reduced formulation that helps to reduce the amount of serum required for cell culture, while still providing the necessary nutrients and growth factors for cell proliferation.
7
Anti halotag by Promega
Sourced in United States
The Anti-HaloTag is a laboratory tool used to detect and study HaloTag-labeled proteins. It functions by binding to the HaloTag protein, allowing for the visualization and isolation of HaloTag-fusion proteins. The Anti-HaloTag provides a reliable method for researchers to investigate protein interactions and localization within cellular systems.
8
Halotag alexa fluor 488 ligand by Promega
Sourced in United States
The HaloTag Alexa Fluor 488 Ligand is a fluorescent labeling agent designed for use with the HaloTag protein fusion system. It provides a covalent and specific labeling of HaloTag-fusion proteins, enabling detection and visualization of the tagged proteins in various applications.
9
Janeliafluor 646 halotag ligand by Promega
Sourced in United States
JaneliaFluor 646 HaloTag ligand is a fluorescent dye that can be used to label HaloTag fusion proteins. The ligand binds covalently to the HaloTag protein, allowing for the visualization and detection of the tagged protein of interest.

More about "HaloTag"

HaloTag is an innovative protein tag technology that enables precise labeling, visualization, and manipulation of target proteins in live cells.
This versatile tool leverages the power of artificial intelligence through PubCompare.ai, a platform that streamlines HaloTag experiments by intelligently comparing and locating the best protocols from literature, preprints, and patents.
HaloTag technology works by covalently binding a synthetic ligand, such as the HaloTag TMR ligand, to the target protein, allowing for its precise tracking and control.
Transfection reagents like Lipofectamine 2000, Lipofectamine 3000, and FuGENE HD can be used to efficiently deliver the HaloTag construct into cells, while Opti-MEM media can optimize transfection conditions.
The HaloTag system is further enhanced by accessories like Magne® HaloTag® Beads, which facilitate the purification and enrichment of HaloTag-labeled proteins, and the Anti-HaloTag antibody, which enables immunodetection of HaloTag-tagged proteins.
Additionally, fluorescent HaloTag ligands, such as the HaloTag Alexa Fluor 488 Ligand and the JaneliaFluor 646 HaloTag ligand, allow for direct visualization and tracking of HaloTag-labeled proteins in live cells.
By leveraging the power of PubCompare.ai, researchers can streamline their HaloTag experiments, ensuring reproducible and accurate results, and ultimately unlock the full potential of this versatile technology.
Whether you're studying protein dynamics, conducting cellular imaging, or exploring protein-protein interactions, the HaloTag system, combined with the AI-driven optimization of PubCompare.ai, can help you achieve maximum experimental results.