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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.
Part number: PT8HALOZEO
List price: USD 2,098.00
Your price:
USD 2,098.00
USD 2,098.00 /each

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

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.

GPCR how it works step 1 plasmid creation receptor cloning pt8halozeo
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.

GPCR how it works step 3 receptor labeling chemical reaction pt8halozeo

 

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.

GPCR how it works step 4 understand the assay principle receptor binding pt8halozeo

 

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.

GPCR how it works step 5 saturation binding kd pt8halozeo
How it works pt8 halotag zeocin 1
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.

GPCR how it works step 6 competitive binding ki pt8halozeo
how it works pt8 halotag zeocin 2

Resources

1-1 of 1 Resources
Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

SDS, COAs, Manuals and more Illuminator

SDS, COAs, Manuals and more

Are you looking for technical documents for this product. We have housed them in a dedicated section., click on the links below to explore.

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