• Introduction
• Applications
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OmicsLink™ ORF Expression Clones with HaloTag®

Applications:

Protein Localization:

Using either membrane permeable (TMR, diAcFAM, Coumarin and Oregon Green) or impermeant (Alexa Fluor 488) HaloTag® ligands, one can label intracellular and membrane proteins, respectively. Subsequently, the labeled proteins can be visualized with a fluorescent microscope (Fig. 3). Cell-impermeant fluorescent dyes are particularly useful in studies of plasma membrane protein characteristics when the HaloTag® fusion protein is engineered to be expressed on cellular membrane.

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

Protein Co-Localization:

If HaloTag®-fusion protein expressing cells are fixed after ligand is added, the labeled cells can be further processed with an antibody against another protein in an immunocytochemistry (IC) assay to determine protein co-localization (Fig. 4). Anti-HaloTag® polyclonal antibody can also be used together with another antibody for co-localization study.

Fig. 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 1µg/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 (Fig. 5). Please refer to Promega’s manual TM260 for this application.

Fig. 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 (Fig. 6).

Fig. 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 5µM 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.

ORF Expression Clones are also available in more than 80 other expression vector systems with different promoters and various N- or C-terminal tags: SNAP, His, GST, MBP, GFP, YFP, AviTag™, HA, Flag, SUMO, IRES and more.