Tag-lite® Technology

Tag-lite® is a non-radioactive, cellular platform that enables the investigation of natural ligand,small molecule or antibody binding to cell surface receptors.

Tag-lite is highly advantageous for GPCR and RTK investigations, as well as biotherapeutic antibody development.

This technology combining HTRF with SNAP-tag®, CLIP-tag® and HaloTag® technologies, offers an alternative to existing cellular bioluminescence resonance energy transfer (BRET) and FRET assays, providing a new targeted way to label your protein of interest.

Contactus

Key features

  • Non-radioactive
  • Homegeneous & filtration-free
  • Ready-to-use kits and reagents for over 42 GPCRs
  • Peer-reviewed, validated technology

Applications

  • Receptor binding assays
  • Screening and profiling of biologics and large molecules

Workflow

1. Construction of plasmid encoding the cell surface protein of interest fused to a SNAP-tag, CLIP-tag or HaloTag.

Cisbio offers SNAP-tag, CLIP-tag, and HaloTag expression constructs for cloning your cell surface protein of interest. We also have has an extensive collection of pre-cloned expression constructs for tagged GPCRs and RTKs, and Cisbio can develop custom ones.

2. Transfection of cells and expression of the tagged cell surface protein.

Tag-lite is compatible with transiently or stably transfected cells. Cisbio offers a variety of stable cell lines and ready-to-use frozen cells which express specific tagged-GPCRs.

3. Labeling of tagged cell surface protein using tag-specific substrates conjugated to an HTRF fluorophore.

Cisbio offers SNAP-tag, CLIP-tag and HaloTag specific substrates labeled with Lumi4-Terbium, green or red acceptor fluorophores.

4. Labeling of the binding partner of interest with the corresponding HTRF fluorophore.

Tag-lite has been used for peptidic, non-peptidic and complex ligand binding as well as antibody binding. Cisbio offers a range of HTRF-labeled ligands for immediate use, and provides custom labeling of ligands as part of its service offering.

5. Conducting biomolecular binding assays

Incubation of labeled cells or cell membrane preparations with binding partners - antibodies, compounds - followed by HTRF measurement.

Tags

All three of these protein fusion tags form covalent bonds with specific chemical substrates upon their intrinsic enzymatic activity. By conjugating HTRF fluorophore to SNAP-tag, CLIPtag or HaloTag substrates, fusion proteins can be linked covalently to the fluorophore post expression in living cells.

  • SNAP-tag and CLIP-tag, are small fusion tags from NEB technology that interact covalently with dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker.
  • HaloTag, a Promega technology, is another orthogonal system for labeling proteins in living cells and in vitro. The HaloTag protein is a 33 kDa enzyme derivatized from a prokaryotic hydrolase

As shown in this diagram, SNAP-tag, CLIP-tag and HaloTag can be fused easily to either the N- or the C-terminal position on proteins of interest, and can then be labeled specifically and covalently with their defined substrates.

Fluorophores & FRET

SNAP-tag labeling once the protein of interest is expressed as a fusion protein (SNAP-tag, CLIP-tag or HaloTag), an HTRF fluorophore is covalently linked to the tag with a tag-specific substrate. Tag-lite substrates are composed of an HTRF fluorophore in combination with the tag-specific linker.

A selection of three substrates is available for each SNAP-tag, CLIP-tagand HaloTag. These substrates are labeled with HTRF fluorophores: terbium cryptate donor (Lumi4-Tb), green or red HTRF acceptors.

These substrates are not cell membrane permeable and are chemically inert towards other proteins, and thus non-specific labeling of other cell surface proteins is not observed. Therefore, only cell surface proteins such as GPCRs can be investigated

SNAP-tag, CLIP-tagor HaloTag are suicide enzymes that react in a very specific way with their respective substrates. These enzymes metabolize their substrate and transfer part of this dye-coupled substrate to themselves. At the end of this irreversible complete process, the enzyme is no longer active, and the remaining tag becomes labeled with the dye. The fused protein labeled either to a donor or to an acceptor is ready to be combined with the other dye partner, and to get the new HTRF assay up and running.

Like all HTRF assays, Tag-lite assays are homogeneous in nature and measured via dual wavelength donor and acceptor emission readouts. If the HTRF-labeled cell surface protein and binding partner interact, FRET occurs and HTRF emissions are produced at both the donor and acceptor wavelengths. In the absence of binding, only HTRF donor emissions are produced.

SNAP-Tag does not affect the receptor pharmacology.

Reverse transfection of vasopressin 1a receptor was performed with the wild type (WT-V1a) and the SNAP-tag V1a (ST-V1a) of the receptor. Inositol 1-phosphate production induced by vasopressin stimulation was assessed using the IP-One kit on the two different cellular models.

Vasopressin EC50s measured with IP1 accumulation assay were very similar on both WT-V1a and ST-V1a confirming that SNAP-tag fusion on the GPCR N-terminal and its specific labeling with the appropriate fluorescent substrate do not affect either the GPCR binding of a specific ligand or the GPCR function.