HTRF Tag-Lite pT8-HaloTag Plasmid, 10µg

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HTRF Tag-Lite pT8-HaloTag Plasmid, 10µg

Tag-lite plasmid featuring a HALO-Tag sequence, a T8 peptide sequence, and a Zeocin resistance gene.Cloning and expression of the GPCR of interest as a SNAP-Tag or CLIP-Tag fusion protein enables its subsequent labeling with Terbium.

For research use only. Not for use in diagnostic procedures.

Product information

SDS, COAs, Manuals and more

Product information

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. PT8HALOZEO is a backbone containing the gene for the HALO tag, a promoter a zeomycin resistance gene and MCS (Multiple Cloning Site).

Specifications

Other Specifications

Assay Points	10
Assay Target Type	Plasmids
Assay Technology	HTRF
Brand	Tag-Lite
Quantity	1
Shelf Life	3650.0 Day(s)
Shipping Conditions	Shipped in Dry Ice
Therapeutic Area	Cardiovascular Infectious Diseases Metabolism/Diabetes NASH/Fibrosis Neuroscience Oncology & Inflammation Rare Diseases
Unit Size	10 mg

Video gallery

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.