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

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

Tag-lite plasmid featuring a SNAP-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.

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Feature	Specification
Application	Receptor-Ligand Binding

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

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Product Variant

Unit Size: 10 ml

Part #:

PT8SNAPZEO

List Price

USD 2,098.00

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.

PT8SNAPZEO is a backbone containing the gene for the SNAP, a promoter, a zeomycin resistance gene and MCS (Multiple Cloning Site).

Specifications

Other 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

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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.