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Tag-lite T8-CLIP (Hygromycin) plasmid HTRF®

Tag-lite plasmid featuring a CLIP-Tag sequence, a T8 peptide sequence, and a Hygromycin resistance gene.Cloning and expression of the GPCR of interest as a SNAP-Tag or CLIP-Tag fusion protein enables its subsequent labeling with Terbium.
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Tag-lite plasmid featuring a CLIP-Tag sequence, a T8 peptide sequence, and a Hygromycin resistance gene.Cloning and expression of the GPCR of interest as a SNAP-Tag or CLIP-Tag fusion protein enables its subsequent labeling with Terbium.
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In stock

Overview

Over the past few years, SNAP-Tag technology combined with TR-FRET has paved the road to the development of many non-radioactive, no-wash binding assays. The method is based on transfecting cells using plasmids encoding a SNAP-Tag and subsequently labeling them with Terbium. PT8CLIPHYG is a backbone containing the gene for the CLIP tag, a promoter, a hygromycin resistance gene and MCS (Multiple Cloning Site).

Benefits

  • LARGE OFFER OF SELECTION MARKERS
  • ANTIBIOTIC RESISTANCE GENE
  • FEATURES A CMV PROMOTER

Step 1 - Plasmid creation

Using standard cloning techniques, insert the GPCR gene of interest into the empty plasmid. Remember that when you are designing a plasmid, the GPCR gene should be kept in the frame.
Diagram of receptor cloning

Step 2 - Plasmid transfection

Use standard transfection techniques (see transient transfection protocol) to transiently express the SNAP-GPCR of interest in your cell line.

Step 3 - Receptor labeling

CLIP-tag®, is a small fusion tag that covalently interacts with specific substrates. CLIP-tag allows specific and covalent labeling of any protein of interest. Cells are provided unlabeled and need to be labeled with Lumi4-Terbium prior to running a binding assay. Labeling reagents are available from the Cisbio catalog in 4 different sizes. For more details see the labeling procedure.
Diagram of a chemical reaction between SNAP-tag and its substrate

Watch this video describing how to label cell surface receptors using Tag-lite® technology.

Step 4 - Understand the assay principle

Running a receptor binding assay using Tag-lite is as easy as it can get. Simply dispense 10 µL of labeled cells into each well, followed by 5 µL of labeled ligand and 5 µL of the compound you wish to test. Like all HTRF assays, Tag-lite assays do not require any washing steps. A diagram of the procedure to be followed is given on the right.
Diagram of a receptor binding assay using the Tag-lite protocol

Step 5 - Saturation binding (KD)

A saturation binding assay measures total and non-specific binding for increasing concentrations of ligand under equilibrium conditions. To perform the assay, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells and then incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding.
Diagram of a saturation binding assay using Tag-lite
Representative data obtained when running a saturation binding assay

Watch this video explaining how to run a saturation binding assay using Tag-lite.

Step 6 - Competitive binding (KI)

A competitive binding assay is performed to measure the dissociation constant, Ki. To perform the assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.
Diagram of a competitive binding assay using Tag-lite
Representative data obtained when running a competitive binding assay

Watch this video explaining how to run a competitive binding assay using Tag-lite.

Evaluation of a Tag-lite binding assay for a class B receptor

In collaboration with Boehringer Ingelheim - Scientific Presentations

Ultra HTS at Bayer: use of IP-One and Tag-lite assays in GPCR drug discovery

In collaboration with Bayer - Scientific Presentations

Tag-lite binding assays

The easy way to approach biology without radioactivity - Flyers

Understanding GPCRs is the key to improved DD

In collaboration with GEN - Guides

Tag-lite, the easiest solution for studying GPCR-ligands interactions

The gold standard technology for receptor binding studies - Videos

Transient Transfection Procedure

Cell transfection protocol - Product Insert

Labeling procedure Tag-lite

Cell surface receptor labeling - Product Insert

HTRF Product Catalog 2020 July update

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Easy method for kinetic binding determination

How to revolutionize your kinetic binding demonstration with HTRF kinase binding platform assays - Application Notes

GPCRs Pharmacology

A comprehensive overview of pharmacology's main ligands - Guides

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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