Revvity, PT8HALONEO, Tag-lite pT8-HaloTag (Neomycin) Plasmid, 10 µg

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. PT8HALONEO is a backbone containing the gene for the HALO tag, a promoter a neomycin resistance gene and MCS (Multiple Cloning Site).
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 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.

 

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
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. 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. For more details see the labeling procedure.

 

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.

 

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.

 

 

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. 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-10 mg