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SNAP tag HTR4 Serotonin R cloned plasmid HTRF®

The Tag-lite HTR4 Serotonin receptor plasmid is used to transiently or stably transfect cells in order to develop an A1 Adenosine receptor binding assay.
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The Tag-lite HTR4 Serotonin receptor plasmid is used to transiently or stably transfect cells in order to develop an A1 Adenosine receptor binding assay.
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Overview

Over the past few years, SNAP-Tag technology combined with TR-FRET has paved the way for 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. Cisbio offers a large collection of such plasmids. All GPCR genes are cloned in an expression vector directly downstream from a CMV promoter, and are provided ready for protein expression and labeling.

All information on this page pertains to the Tag-lite plasmid cloned with the HTR4 Serotonin receptor.

Benefits

  • FULL LENGTH RECEPTOR
  • HIGH EXPRESSION SYSTEM
  • VALIDATED IN EXPRESSION

Step 1 - Plasmid transfection

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

Step 2 - Receptor labeling

SNAP-tag® is a small fusion tag that covalently interacts with specific substrates. It enables the specific and covalent labeling of any protein of interest (refer to labeling procedure). Cells are provided unlabeled, and need to be labeled with Lumi4-Terbium prior to running a binding assay. Labeling reagents are available in 4 different pack sizes from the Cisbio catalog.
Step 2 - Receptor labeling
Watch this video describing how to label cell surface receptors using Tag-lite® technology.

Step 3 - 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.
Step 3 – Understand the assay principle

Step 4- 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 set amount of labeled cells, and then incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding.
Step 4 – Saturation binding (KD)
Step 4 – Saturation binding (KD)
Watch this video explaining how to run a saturation binding assay using Tag-lite.

Step 5 - Competitive binding (KI)

A competitive binding assay is performed to measure the dissociation constant, Ki. To perform this assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.
Step 5 – Competitive binding (KI)
Step 5 – Competitive binding (KI)
Watch this video explaining how to run a competitive binding assay using Tag-lite.

Transient Transfection Procedure

Cell transfection protocol - Product Insert

Labeling procedure Tag-lite

Cell surface receptor labeling - Product Insert

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Plate Reader Requirement

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