HaloTag® 7 Technology — More versatile than most other tags

Figure 1. OmicsLink ORF cDNA expression clones with N- and C- HaloTag® in various mammalian vector systems.

How it works

Figure 2. Covalent and specific binding of a variety of synthetic reporter and affinity ligands to HaloTag® proteins allows detection, affinity-binding or solid-phase fixation of proteins of interest.

Figure 3. Live cells expressing HaloTag® fusion protein labeled with three different ligands. HeLa cells transiently transfected with HaloTag® pHT2 expression clones were labeled with 5 μM HaloTag® TMR Ligand (Panel A); 10μM HaloTag® diAcFAM Ligand (Panel B); or 25 μM HaloTag® Coumarin Ligand (Panel C).

Protein Co-Localization

If HaloTag® fusion-protein expressing cells are fixed after the ligand is added, the labeled cells can be further processed with an antibody against another protein in an immunocytochemistry assay to determine protein co-localization (Figure 2). Anti- HaloTag® polyclonal antibody can also be used together with another antibody for co-localization study.

Figure 4. Fixed cells expressing p65-HaloTag® fusion protein labeled with HaloTag® TMR ligand and an antibody to tubulin. HeLa cells transiently transfected with the p65-HaloTag® were stained with TMR ligand for 15 minutes at 37^.C. Cells were then fixed and exposed to mouse Anti-βIII tubulin antibody at 1 μg/ml followed by incubation with Alexa Fluor 488-conjugated secondary. Panel A: TMR fluorescence. Panel B. Alexa Fluor-488 fluorescence. Panel C: Overlaid Alexa Fluor™-488 and TMR fluorescence and transmitted light. Panel D: Overlaid AlexaFluor-488 and TMR fluorescence. Please refer to Promega’s manual TM260 for this application.

Protein-DNA interaction

Immobilized ligands (such as HaloLink™ Resin) can be used to precipitate HaloTag® fusion proteins cross-linked to DNA. This protocol abolishes the need for antibodies in classic chromatin immunoprecipitation (ChIP) assays and enables experiments in the absence of highly functional antibodies. Please refer to Promega’s manual #TM075 for this application.

Multiplex pulse-chase labeling for protein trafficking studies

Using different fluorescent ligands, users can label HaloTag® fusion protein of interest at different time intervals in live cells so that protein trafficking can be monitored over time (Figure 3). Please refer to Promega's manual TM260 for this application.

Figure 5. Spatial and temporal separation of proteins using HaloTag® technology. Twelve hours after cell labeling, imaging showed the differentially labeled proteins as they moved from the cytoplasm to the membrane and as they internalized from the membrane. Detailed description of the experimental design and results can be found in Svendsen, S., et al., (Promega Notes 95 (2007) 16–19).

Protein enrichment, SDS-PAGE and western blot analysis

Using biotinylated ligand with immobilized strepavidin, users can enrich HaloTag® fusion proteins of interest for subsequent protein purification, enzymatic study, mass spectrometry analysis, or post-translational modification. Furthermore, after labeling with fluorescent ligand, HaloTag® fusion proteins can be separated by SDS-PAGE, fluoroimaged directly, and/or transferred for western blot analysis (Figure 4).

Figure 6. Fast, efficient and highly specific labeling of the HaloTag® proteins expressed in mammalian cells. CHO-K1 control cells (lanes 1–6) or cells transiently transfected with HaloTag® pHT2 expression clone (lanes 7–12) were labeled with 5 μM HaloTag® TMR Ligand for different periods of time at 37°C (0.5, 1, 2, 5, 15 and 30 minutes). Proteins were resolved by SDS-PAGE and analyzed on a Hitachi FMBIO® fluorescence scanner.

This product is manufactured under licensing agreement with Promega Corporation. HaloTag® is a trademark of Promega Corporation. All figures in this page are adapted from Promega with permission.