Description
MAb Anti FLAG-d2 is an antibody raised against FLAG® fusion proteins labeled with d2. The anti-FLAG antibody will recognize the FLAG sequence at the N-terminus, intra, or C-terminus of FLAG fusion proteins.
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, and receptor/ligand.
HTRF can detect a broad range of affinity constants ranging from picomolar to low millimolar.
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 resulting signal is proportional to the binding of the 2 partners. In the example shown here, MAb Anti FLAG-d2 binds to the FLAG tagged partner A, while partner B* binds to a specific Ab labeled with an HTRF donor.
*partner B can also be biotinylated, tagged, or Fc fused. In these cases, use the corresponding HTRF reagent (anti-Tag, anti-species, protA, Streptavidin) labeled with a donor for the detection.
Assay protocol
The example on the right describes the protocol using a 20 µL final assay volume for the detection of an interaction between a FLAG-tagged partner A and a non-tagged partner B*. Dispense the 2 partners (10 µL), incubate, add MAb Anti FLAG-d2 (5 µL) and anti-partner B labeled with donor (5 µL), then 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 a donor for the detection.
Build HTRF interaction assays for your specific application
Reagents are sold by the number of tests (20 µL reactions). Antibodies conjugated to XL665 or d2 are 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 the active moiety?
The average conjugate quantity per well reflects overall biological material content. Use of 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.
Recommended quantities of Cryptate and XL665 conjugates
The amount of cryptate conjugate must not be excessive, in order to avoid 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.
