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HaloTag® 7 Technology — More versatile than most other tags

Introduction

The HaloTag® is the preferred system for multicolor cell imaging experiments with either live or fixed cells. This multi-functional tag binds covalently and specifically to a variety of synthetic ligands enabling tagged proteins to be labeled with fluorophores for both in vitro and in vivo imaging or with affinity agents for purification.

HaloTag® also enhances protein expression and solubility in addition to facilitating efficient protein purification.Thus, the HaloTag® can be used for a variety of downstream applications.

 

Technology

The HaloTag® protein, a genetically engineered derivative of a dehalogenase, efficiently forms a covalent bond with various synthetic HaloTag® ligands. The 34 kDa monomeric protein can be fused at either the N-or C-terminus to proteins of interest and enables expression in both prokaryotic (E. coli) and various eukaryotic cells.

 

Combined with OmicsLink™ expression-ready clones

GeneCopoeia offers HaloTag® technology in a variety of mammalian and lentiviral expression vectors with the CMV promoter and neomycin selection marker. A collection of 20,000 human and 15,000 mouse genes may be ordered with HaloTag®.

 

Specific

Covalent and specific binding of a variety of synthetic reporter and affinity ligands to HaloTag® fusion proteins allows detection and affinity-binding or solid-phase fixation of tagged proteins.See the list of reporter and affinity ligands available from GeneCopoeia.

 

Rigorous quality control and assurance process

All ORF cDNA clones in the OmicsLink collection were generated from sequence validated full-length cDNA clones or high-quality human tissue cDNA libraries to construct expression-ready clones.

Once the clone is constructed, GeneCopoeia follows additional quality control processes to ensure the right clone is delivered.

  • All ORF cDNA clones are fully sequenced.
  • PCR amplification and size validation
  • Enzyme digestion check of the integrity of whole plasmid.

Vectors

List of expression vectors with HaloTag® technology

Vector Promoter  Host cell Selection marker Tag Protease site
pReceiver-Lv64 CMV Stem/primary cell Neomycin N-HaloTag Tev protease
pReceiver-Lv65 CMV Stem/ primary cell Neomycin C-HaloTag Tev protease
pReceiver-Lv110 CMV Stem/primary cell Puromycin N-HaloTag Tev protease
pReceiver-Lv129 CMV Stem/primary cell Puromycin C-HaloTag Tev protease
pReceiver-Lv211 CMV Stem/primary cell Hygromycin C-Halo-IRES-Hygromycin N/A
pReceiver-M49 CMV Mammalian Neomycin N-HaloTag Tev protease
pReceiver-M50 CMV Mammalian Neomycin C-HaloTag Tev protease
pReceiver-B80 T7 E. Coli N/A N-Halo Tev protease

 

 

HaloTag ORF cDNA clones

 

HaloTag ORF cDNA clones

 

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

 

How it works

Labeling of HaloTag proteins

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.

Applications and Data

Application of HaloTag® technology

Protein Localization

Using either membrane permeable or impermeant HaloTag® ligands, intracellular and membrane proteins can be labeled. These labeled proteins can be visualized with a fluorescent microscope (Figure 1). 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.

   
Live cells expressing HaloTag® fusion protein labeled with three different ligands  

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.

   
Fixed cells expressing p65-HaloTag® fusion protein labeled with HaloTag® TMR ligand and an antibody to tubulin  

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.

   
Spatial and temporal separation of proteins using HaloTag® technology  

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).

   
Fast, efficient and highly specific labeling of the HaloTag® proteins expressed in mammalian cells  

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.

Other products

HaloTag related products can be purchased directly from Promega

Catalog #  Ligand  Excitation Maximum  Emission Maximum  Application
G2801 HaloTag® Oregon Green® Ligand 496nm 516nm Intracellular protein labeling
G1001 HaloTag® Alexa Fluor™ 488 Ligand 494nm 517nm surface membrane protein labeling
G8281 HaloTag® Biotin     Protein interaction and/or enrichment
G8591 HaloTag® PEG-Biotin     Protein enrichment
G1911 HaloLink™ Resin     ChIP Assay

 

Learn more about the expression-ready ORF cDNA clone collection available with various other fusion tags. in bacterial, lentiviral, mammalian, insect, yeast and wheat germ cell free systems.

Please visit www.promega.com for more information on HaloTag® related products.

This product is manufactured under licensing agreement with Promega Corporation. HaloTag® is a trademark of Promega Corporation.