SNAP tag 5HT1A 5-Hydroxytryptamine 1AR cloned plasmid

The Tag-lite 5HT1A 5-Hydroxytryptamine 1A plasmid is used to transiently or stably transfect cells for the purpose of developing a 5HT1A 5-Hydroxytryptamine 1Areceptor binding assay.
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The Tag-lite 5HT1A 5-Hydroxytryptamine 1A plasmid is used to transiently or stably transfect cells for the purpose of developing a 5HT1A 5-Hydroxytryptamine 1Areceptor 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 5HT1A 5-Hydroxytryptamine 1A receptor.

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. SNAP-tag allows 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 from the Cisbio catalog in 4 different sizes.

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 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 fixed amount of labeled cells and then incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding.

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 the assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.

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

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Understanding GPCRs is the key to improved DD

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

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Transient Transfection Procedure

Cell transfection protocol - Product Insert

Labeling procedure Tag-lite

Cell surface receptor labeling - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

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Safety Data Sheet pSNAP-5HT1A-R / PSNAP5HT1A

PSNAP5HT1A - Safety Data Sheet

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