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.
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®.
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.
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|
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.
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.
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).
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.
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.
Reporter and Affinity HaloTag® Ligands
HaloTag® ligands are small molecules capable of covalently binding the HaloTag® protein in live cells or in vitro. These ligands are composed of two critical components including a common HaloTag® reactive linker that binds to the HaloTag® protein and a functional group such as fluorescent dye or affinity agent.
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.
List of HaloTag® ligands and Anti-HaloTag antibody
|Buy||Catalog#||Ligand||Unit||Qty||Excitation Maximum||Emission Maximum||Application||Price ($USD)|
|G8272||HaloTag® diAcFAM Ligand||30μl at
|1||494nm||526nm||Intracellular protein labeling||489|
|G8251||HaloTag® TMR Ligand||30μl at
|1||555nm||585nm||Intracellular protein labeling||489|
|G8581||HaloTag® Coumarin Ligand||30μl at
|1||353nm||434nm||Intracellular protein labeling||475|
|G9281*||Anti HaloTag® pAB||200μg, 1mg/ml||1||Labeling after fixing or protein enrichment||225|
*G9281 can only be ordered together with ORF expression clones.
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.