Description
Strep-Tactin has been labeled with Tb crypate. 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.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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, Strep-Tactin®-Tb binds to the Strep-Tag®II tagged partner A, while partner B* binds to a specific Ab labeled with an HTRF acceptor. *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 an acceptor for the detection.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume for detection of 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®-Tb cryptate (5 µL) and anti-partner B labeled with acceptor (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 an acceptor for the detection.
How do the number of tests relate to the active moiety?
The average conjugate quantity per well reflects the 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 total quantity of 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.
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.
