HaloTag Technology

One powerful technology – Multiple applications for protein functional analysis

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The HaloTag® Technology provides new options to enable the understanding of protein function in a biochemical and cellular environment. The technology is based on the efficient formation of a covalent bond between a protein fusion tag and synthesis ligands. These ligands enable researchers to perform a variety of protein analysis applications.

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  • Technology
    Advantages
  • Protein:protein
    Interactions
  • Protein:DNA
    Interactions
  • In vivo
    Localization

Technology Advantages

HaloTag Technology

 

 

 

 

 

 

 

 

  • Design only one construct for multiple applications: Obtain new functionality by using a different HaloTag ligand without having to design and clone a new expression construct.
  • Minimize nonspecific binding: Covalent attachment to HaloTag ligand or ligand-coated surface enables stringent washing, minimizing nonspecific background.
  • Enhance the detection of protein:protein interactions: Fast binding kinetics enable binding of proteins at low concentrations while allowing detection of rare events.

 

Complete Functional Protein Characterization
Using a vector based on the HaloTag Technology enables downstream applications such as cell imaging, immobilization and analysis of protein activity without having to reclone into specific vectors.

Protein:Protein Interactions

Determining the protein:protein interaction map of the whole proteome is one major focus of functional proteomics.

 

Analyze protein:protein interactions in pull-down assays

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Pull-down assays can be used to probe interactions between a HaloTag fusion protein (bait) and potential interacting partners (prey) in solution. Bait-prey complexes are isolated from solution by specific binding of the bait to HaloLink Resin, which consists of a HaloTag ligand linked to a Sepharose surface.

The covalent and oriented attachment of fusion proteins to HaloLink Resin provides an excellent choice for detection of protein interactions using the pull-down method. HaloLink Resin is predominantly intended for detection of protein interactions when both protein partners are expressed in the in vitro expression systems or for isolation of protein complexes from mammalian cell in vivo.

 

Advantages:

  • Low nonspecific binding: Covalent attachment to ligand or ligand-coated surface enables stringent washing, minimizing nonspecific background without dissociation of bound HaloTag fusion proteins.
  • Enhanced detection of protein:protein interactions: HaloTag Protein binds the resin rapidly with high affinity, allowing efficient immobilization of proteins present at low concentrations.
  • Efficient binding of proteins without purification or protein modification prior to immobilization: Immobilization of proteins onto HaloLink Resin is covalent, rapid and selective.

 

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Detection of enzymatic activity

Immobilization of enzymes and study of their activities can be achieved with the HaloTag technology. Covalent attachment of proteins to the HaloLink Resin allows the assay of enzymatic activities over a long period of time in a variety of buffer conditions without protein dissociation from the resin. Other affinity purification resin tags are often used to attach enzymes to the surface; however, after incubation fusion proteins will dissociate from the resin. Because HaloTag fusion proteins are covalently bound to the HaloLink Resin, dissociation does not occur.

 

Advantages:

  • Maximal enzyme activity of bound protein: Oriented immobilization of HaloTag fusion protein.
  • Minimize nonspecific binding: Covalent attachment to ligand or ligand-coated surface enables stringent washing, minimizing nonspecific background without dissociation of bound HaloTag fusion proteins.
  • Enhanced detection of protein:protein interactions: HaloTag Protein binds the resin rapidly with high affinity, allowing efficient immobilization of proteins present at low concentrations.

 

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Purification of Nontagged Protein

Release of the protein of interest from the HaloLink Resin is possible with TEV protease. All HaloTag CMV Flexi Vectors contain a protease cleavage site for TEV situated in the linker sequence between the HaloTag Protein and the protein of interest. Because the cleavage is performed on the protein bound to the resin, any contaminating proteins can be removed by extensive washing before the cleavage reaction, and the released target protein is tag-free.

 

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Products

Ligand for Protein Immobilization

 

HaloTag Vectors

HaloTag Vectors for Mammalian Protein Expression

HaloTag Vectors for E. coli Protein Expression

HaloTag Vectors for Cell-Free Protein Expression

 

HaloTag® pHT2 Vector

 

Related Products

 

Protocol

 

Publications/Literature

 

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Protein:DNA Interactions

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The HaloCHIP™ System is a novel method designed for the covalent capture of intracellular protein:DNA complexes without the use of antibodies and offers an efficient and robust alternative to the standard chromatin immunoprecipitation (ChIP) method.

 

Advantages

  • No requirement for antibody: No need to make your own or purchase expensive, qualified antibodies.
  • Obtain results faster: Obtain data in 24 hours with fewer steps to minimize potential experimental errors.
  • Improved signal-to-noise ratio: Enables detection of small changes in protein binding patterns using a minimal number of cells.

 

 

Products

HaloCHIP™ System

 

HaloTag Vectors for Mammalian Protein Expression

 

Related Products

 

Protocol

 

Publications/Literature

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In vivo Localization

The HaloTag Technology provides new options for rapid, site-specific labeling of proteins in living cells. The ability to create labeled HaloTag fusion proteins with a wide range of optical properties and functions enables the imaging and localization of labeled HaloTag protein fusions in live-or fixed-cell populations.

 

Imaging-based pulse-chase experiments & temporal/spatial seperation in live cells

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The HaloTag technology permits tethering of fluorophores with different wavelengths to a reporter protein using a single genetic construct. Fluorophores attached to HaloTag ligands with different abilities to cross the cell membrane can be used to regionally label a protein of interest (see image in brochure).

 

Advantages:

  • Multiple applications with one construct: Obtain new functionality by using a different HaloTag ligand. No need to design and clone a new expression construct.
  • Live- or fixed-cell imaging: The stable covalent bond allows imaging of fixed cells and analysis of the labeled protein under stringent conditions.
  • High specificity of protein fusion: HaloTag ligands specifically bind to the HaloTag protein fusion but not to other proteins in mammalian cells.
  • Fast cell-to-gel analysis: Covalent bond enables cells to be imaged, lysed and fusion protein analyzed on an SDS gel.

 

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Perform multiplexing in live-cell imaging experiments – combine with an IFP (intrinsically fluorescent protein)

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The HaloTag Technology can simplify multicolor experiments. Since the HaloTag Protein is not an IFP, the choice of fluorescent labels, including secondary and tertiary fluorophores, can be made after creating the HaloTag fusion protein. This feature allows more flexibility in the choice of fluorophores.

 

Advantages:

  • Multiple applications with one construct: Obtain new functionality by using a different HaloTag ligand. No need to design and clone a new expression construct.
  • High specificity of protein fusion: HaloTag ligands specifically bind to the HaloTag protein fusion but not to other proteins in mammalian cells.
  • More flexibility in the choice of fluorophores: The choice of secondary and tertiary fluorescent labels can be made after creating the HaloTag fusion protein.

 

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HaloTag ligands retain their fluorescent properties after fixation, allowing the possibility of performing multiplexing experiments that employ immunocytochemistry (ICC).

 

Advantages:

  • Multiple applications with one construct: Obtain new functionality by using a different HaloTag Lignad. No need to design and clone a new expression construct.
  • Live- or fixed-cell imaging: The stable covalent bond allows imaging of fixed cells and analysis of the labeled protein under stringent conditions.
  • High specificity of protein fusion: HaloTag ligands specifically bind to the HaloTag Protein Fusion but not to other proteins in mammalian cells.
  • Fast cell-to-gel analysis: Covalent bond enables cells to be imaged, lysed and fusion protein analyzed on an SDS gel.

 

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Cell-to-gel analysis of protein

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The HaloTag Protein-Ligand interaction is stable and highly specific. The fluorescently labeled HaloTag Protein can be boiled with SDS-PAGE sample buffer and resolved by SDS-PAGE without detectable loss of the fluorescent signal. In addition, the availability of the Anti-HaloTag pAb provides an additional tool for detection of HaloTag fusion proteins by traditional Western blot applications.

 

 

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Products

Ligands for Cellular Imaging

HaloTag Fluorescent Ligand - Cell Impermeant

HaloTag Fluorescent Ligands - Cell Permeant

 

HaloTag Vectors

HaloTag Vectors for Mammalian Protein Expression

 

HaloTag® pHT2 Vector

 

Related Products

 

Protocols

 

Publications/Literature

 

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