HTRF Strep-Tactin®-XL665 HTRF®
HTRF Strep-Tactin®-XL665 can be used to capture Strep-tag® II and Twin Strep-tag®-tagged proteins.
- High affinity
Strep-Tactin® has been labeled with XL665. Biotin, Twin Strep-tag®,and Strep-tag®II bind to Strep-Tactin® with high affinity. The binding is rapid and stable, making it an ideal choice for use in a variety of assays such as enzyme assays, protein-protein binding assays, and molecular biology assays.
- LARGE COMPLEX DETECTION
- BATCH-TO-BATCH REPRODUCIBILITY
- LOW TO HIGH KD DETECTION
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, Strep-Tactin®-XL665 binds to the Strep-Tag®II 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.
The example on the right describes the protocol using a 20 µL final assay volume for detecting an interaction between a Strep-Tag®II-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add Strep-Tactin®-XL665 (5 µL) and anti-partner B labeled with donor (5 µL), incubate, and then read.
*partner B can also be tagged, Fc fused, or directly labeled. In these cases use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin), labeled with a donor for the detection.
Reagents are sold by the number of tests (20 µL reactions). XL665 or d2 conjugates are supplied on the basis of 40 ng of Strep-Tactin® per well. The amount of active moiety per vial is also indicated (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. Strep-Tactin®).
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 indicated by Cisbio is constant and based on the number of tests ordered.
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.
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Plate Reader Requirement
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