A2BR expressing cells labeled with Terbium
Tag-lite cells transiently expressing the A2B receptor with Terbium
SNAP tag A2B AdenosineR cloned plasmid HTRF®
The Tag-lite A2B Adenosine receptor plasmid is used to transiently or stably transfect cells in order to develop an A2B Adenosine receptor binding assay.
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 Adenosine A2B 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 sizes from the Cisbio catalog.
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.
Transient Transfection Procedure
Cell transfection protocol - Product Insert
Cell surface receptor labeling - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
Product Insert pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Product Insert
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Webinar | Association Rate Constant: The Unsung Hero of Drug Discovery Kinetics
Available On-demand - Videos
Easy method for kinetic binding determination
How to revolutionize your kinetic binding demonstration with HTRF kinase binding platform assays - Application Notes
The gene therapy industry: a brief report
An overall view of the gene therapy industry - Guides
Gene therapy processes: the significant advances, key facts and techniques - Infographics
A comprehensive overview of pharmacology's main ligands - Guides
GPCRs: the pathway to Discovery
Complete solutions for GPCR Drug Discovery - Brochures
Safety Data Sheet (DEU) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (ELL) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (FRA-FR) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (ITA) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (SPA) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (ENG-GB) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Safety Data Sheet (ENG-US) pSNAP-A2B-R / PSNAPA2B
PSNAPA2B - Safety Data Sheet
Plate Reader Requirement
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