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Tag-lite pT8-SNAP (Neomycin) Plasmid, 10 µg

Tag-lite plasmid featuring a SNAP-Tag sequence, a T8 peptide sequence, and a Neomycin 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.

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Feature Specification
Application Receptor-Ligand Binding

Tag-lite plasmid featuring a SNAP-Tag sequence, a T8 peptide sequence, and a Neomycin 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.

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variant
Unit Size: 10 ml
Part #:
PT8SNAPNEO
List Price
USD 2,098.00

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.

PT8SNAPNEO is a backbone containing the gene for the SNAP, a promoter, a neomycin resistance gene and MCS (Multiple Cloning Site).

Specifications

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 ml

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
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
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
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 pt8snapneo
How it works pt8 snap neomycin 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
how it works pt8 snap neomycin 2

 

Resources

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

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