Description
What Experiments Can Be Performed with HaloTag® Ligands?
With a single HaloTag® fusion construct, the HaloTag® ligand determines the function. Image, pull down or analyze your protein with a single construct. As your research evolves, simply change the ligand to change the function.
Live-Cell Fluorescence Imaging: Visualize protein localization and dynamics in real time.
Super-Resolution Microscopy: JF dyes are optimized for STORM, STED and single-molecule imaging, due to their superior photostability and fluorogenic properties.
In Vivo Imaging: Janelia Fluor® in vivo kits (JF552, JF608, JF669) are formulated for use in animal imaging, including brain imaging.
Surface Protein Labeling: Cell-impermeant ligands (JF549i, JF635i, XFD 488i, XFD 660i) selectively label HaloTag® fusion proteins on the cell surface or in the extracellular environment without entering the cell.
Pulse-Chase Labeling: Sequential addition of differently colored ligands tracks protein cohorts over time, enabling turnover and trafficking studies.
Flow Cytometry/FACS: Fluorescent HaloTag® Ligands are compatible with standard FACS instruments across multiple laser lines.
Protein Pull-Downs: The stable covalent bond enables capture of HaloTag® fusion proteins under stringent conditions using HaloTag® Biotin Ligand or HaloLink™ Resin, supporting co-immunoprecipitation, proteomics and interactome studies.
CRISPR Knock-In Tagging: The HaloTag® tag is commonly inserted at endogenous loci by CRISPR/Cas9 to study proteins at native expression levels without artifacts, preserving regulatory elements, expression timing and stoichiometry that overexpression models can't replicate.
Live-Cell Labeling with Cell-Permeant Ligands
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Parental U2OS cells and U2OS cells stably expressing HaloTag® protein fused to three copies of a nuclear localization sequence were adhered to glass-bottom chamber slides and labeled with Janelia Fluor® 503, Janelia Fluor® JFX 554 or Janelia Fluor® 635 HaloTag® Ligand for 30 minutes at 37°C + CO 2 in a cell culture incubator. Cell medium was replaced with a phenol red-free medium. Cells were imaged with 488nm laser excitation for Janelia Fluor® 503 HaloTag® Ligand ( Panel A ), 561nm laser excitation for Janelia Fluor® JFX 554 HaloTag® Ligand ( Panel B ), 637nm laser excitation for Janelia Fluor® 635 HaloTag® Ligand ( Panel C ). In HaloTag-expressing cells, labeling was restricted to the nucleus. Parental cells (without HaloTag) showed no labeling. Images were collected using a Nikon AX/AXR confocal microscope with a plan fluor 40X oil objective.
Live-Cell Labeling with Cell-Impermeant Ligands
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Live-cell labeling of HaloTag® fusion proteins with impermeant fluorescent ligands. U2OS cells stably expressing HaloTag® protein fused to the truncated beta 1 integrin receptor extracellular surface sequence or control U2OS cells expressing HaloTag® protein fused with three copies of a nuclear localization sequence were adhered to glass-bottom 96-well plates and labeled with either Alexa Fluor® 488 ( Panel A ), Alexa Fluor® 660 ( Panel B ), Janelia Fluor® 549i ( Panel C ) or Janelia Fluor® 635i ( Panel D ) HaloTag® Ligand for 30 minutes at 37°C + CO₂ in a cell culture incubator. After labeling, the medium was replaced with phenol red-free medium, and cells were imaged using a Nikon AX/AXR confocal microscope with a 20X objective. Fluorescence was captured using 488nm laser excitation for Alexa Fluor® 488 ( Panel A ), 640nm laser excitation for Alexa Fluor® 660 ( Panel B ), 561nm laser excitation for Janelia Fluor® 549i ( Panel C ) and 640nm laser excitation for Janelia Fluor® 635i ( Panel D ). In HaloTag-expressing cells, labeling was restricted to the membrane, while control U2OS cells expressing nuclear-localized HaloTag showed no labeling.
FACS of Janelia Fluor® HaloTag® Ligand-Labeled Proteins
HEK293 cells were edited using CRISPR/Cas9 to tag endogenous BRD4 protein with HaloTag at the N-terminus. The edited cells pool was labeled with Janelia Fluor® 646 HaloTag® Ligand and sorted on a BD FACSMelody™ sorter to isolate single clones. After expansion of cell populations, HaloTag-expressing clones and parental HEK293 cells labeled with Janelia Fluor® 646 HaloTag® Ligand were analyzed by flow cytometry on a BD LSRFortessa™. Clonal populations expressing HaloTag fused to endogenous BRD4 ( blue histogram ) showed a fivefold shift in mean fluorescence compared to labeled parental HEK293 cells ( pink histogram ).
Use HaloTag® Ligands in Your Protein Analysis
Learn more about HaloTag® technology and why it's a powerful tool for protein labeling and analysis.
