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HTRF Protein A-XL665, 5,000 Assay Points

XL665-labeled Protein A for capturing antibodies in protein/protein interaction assays.

For research use only. Not for use in diagnostic procedures.
Part Number: 61PRAXLA
Unit Size: 5,000 Assay Points
Part Number: 61PRAXLB
Unit Size: 20,000 Assay Points
  • Protein A has been labeled with XL665. Protein A-Eu cryptate may be used to detect the binding of immunoglobulins, most notably IgGs from several mammalian species.

    This reagent can be used in both biochemical and cellular formats to study a wide variety of interactions: protein/protein, protein/peptide, protein/DNA, protein/RNA, protein/carbohydrate, protein/small molecule, receptor/ligand.

    HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.

  • Assay Points
    5000
    Assay Target Type
    Fluorescent reagent
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Cardiovascular
    Infectious Diseases
    Inflammation
    Metabolism/Diabetes
    NASH/Fibrosis
    Neuroscience
    Oncology & Inflammation
    Rare Diseases
    Unit Size
    5,000 Assay Points
  • Assay principle

    In an HTRF interaction assay, one partner is labeled (directly or indirectly) with the donor, and the other with the acceptor (again, directly or indirectly). The intensity of the signal is proportional to the binding of the 2 partners. In the example shown here: Protein A-XL665 binds to the Fc fused tagged partner A while partner B* binds to a specific Ab labeled with an HTRF donor. *partner B can also be biotinylated, tagged, Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with donor for the detection.

    HTRF Protein A-XL665 1.svg

     

    Assay protocol

    The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a Fc fused-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add Protein A-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate and read. *partner B can also be biotinylated, tagged, Fc fused or directly labeled. In these cases, use the corresponding HTRF reagent (anti-Tag, anti species, protA, Streptavidin) labeled with donor for the detection.

    HTRF Protein A-XL665 2.svg

     

  • Build HTRF interaction assays for your specific application

    Reagents are sold by the number of tests (20 µL reactions). XL665 or d2 conjugatesare supplied on the basis of 20 ng of antibody per well. The amount of active moiety per vial is also provided (as well as the number of tracers per vial - see product description sheet). The active moiety is defined as the active part of a conjugate (e.g. antibody).

    How do the number of tests relate to active moiety?

    The average conjugate quantity per well reflects overall biological material content. Using the active moiety amount is generally preferred to the quantity of total conjugate. For Cryptate and d2 conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. This is not the case for XL665 labeled entities for which the quantity of total conjugate will vary depending on the final molar ratio of the XL665 conjugate, however, the amount of active moiety, provided by Revvity, is constant and based on the number of tests ordered. Motif Active Moiety 5,000 tests Europium conjugate XL665 conjugate Protein A 3.5 µg 50 µg.

    HTRF Protein A-XL665 3.svg

     

    Recommended quantities of Cryptate and XL665 conjugates

    Cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a cryptate concentration of 1 to 5nM is appropriate, and will generate 20,000 to 80,000 cps at 620 nm depending on the HTRF compatible reader used. The XL665 conjugate must match its assay counterpart as closely as possible in order for the maximum number of biomolecules to be tagged with the XL665 acceptor. Thus, to detect a tagged molecule at an assay concentration of 20nM, the concentration of anti-Tag-XL665 should be equimolar or higher.

Resources

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Guide
HTRF solutions, guide to major applications

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