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HTRF Tag-Lite pT8-SNAP Plasmid, 10µg

Tag-lite plasmid featuring a SNAP-Tag sequence, a T8 peptide sequence, and a Hygromycin 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: PT8SNAPHYG
Part Number: PT8SNAPHYG
  • 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. PT8SNAPHYG is a backbone containing the gene for the SNAP, a promoter, a hygromycin resistance gene and MCS (Multiple Cloning Site).

  • 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
  • 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 pt8snaphyg
    how-it-works-pt8-snap-hygromycin-1.svg
    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 hygromycin 2

     

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Guide
HTRF solutions, guide to major applications

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