Revvity, PSNAP5HT1A, Tag-lite pSNAP-5HT1A Plasmid, 10 µg

Overview
Over past few years, SNAP-Tag technology combined with TR-FRET has paved way development of many non-radioactive, no-wash, binding assays. method is based on transfecting cells using plasmids encoding a SNAP-Tag and subsequently labeling m with Terbium. Revvity 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 protein expression and labeling.
All information on this page pertains to Tag-lite plasmid cloned with 5HT1A 5-Hydroxytryptamine 1A receptor.
Tag-lite assays offer many features and applications over traditional receptor/ligand binding assays:

Non-radioactive
Homogeneous and filtration-free
Ready-to-use kits and reagents for binding assays
Does not alter the receptor pharmacology
Peer-reviewed, validated technology
Thorough kinetic studies with true Kd and Ki values, association (Kon) and dissociation (Koff) rate constants
Screening and profiling of biologics and large molecules
How it works
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 Revvity catalog in 4 different sizes.

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.

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. Application-Receptor-Ligand Binding
Brand-Tag-lite
Detection Modality-HTRF
Product Group-Plasmids
Shipping Conditions-Shipped in Dry Ice
Target Class-GPCR
Technology-TR-FRET
Therapeutic Area-Cardiovascular Infectious Diseases Metabolism/Diabetes NASH/Fibrosis Neuroscience Oncology & Inflammation Rare Diseases
Unit Size-1 each