HTRF® toolbox reagent usage

A broad range of fluorophore conjugates to increase assay design flexibility. These conjugates are particularly useful in cases where protein-specific reagents are not available.

Essential for studying molecular interactions, tagged biomolecules offer researchers flexibility in assay design. The most frequently used tags consist of protein and peptide structures such as GST, 6HIS, c-myc, FLAG®, HA, and MBP. Small organic motifs, such as biotin and dinitrophenyl (DNP), are also widely used for developing assays. The HTRF reagent toolbox provides a comprehensive selection of conjugated binders – anti-tag antibodies, streptavidins, and lectins – for detecting this broad diversity of motifs. The HTRF reagent toolbox also includes secondary murine, sheep, rabbit, and human antibodies, as well as immunoglobulin binding proteins. Most of these reagents are available as Eu3+ cryptate, Lumi4®-Tb cryptate, XL665, and d2 conjugates. They may also be labeled with other fluorophores by our service teams upon request.

The advantages

Provides a unique set of reagents for developing homogeneous high-throughput assays. Allows flexible and straightforward scale-up for primary and secondary screening phases. Enables multiple target developments and assay configurations, such as protein:protein and nuclear receptor screening, receptor dimerization, protease and investigation of other enzymatic processes.

Features

  • High-affinity monoclonal and polyclonal antibodies
  • High-range, streptavidins and brighter SA-XLent! reagents for assays requiring high sensitivity
  • Resistant to most buffer conditions and additives (e.g. DMSO, pH, chelators, ionic strength)
  • Compatible with membrane- and cell-based assays
  • Lyophilized for easy handling and long-term storage
  • Proven batch-to-batch reproducibility

Principle and definitions

All reagents in the HTRF toolbox are supplied with reference to a low-volume 384-well format for a final assay volume of 20 µL. HTRF is particularly well suited to miniaturization, and the number of wells specified for each product reference can be increased depending on the level of miniaturization. Reference tables also give the average quantities of active moiety per vial. Active moiety is defined as the active part of a conjugate (e.g. antibody, streptavidin), as shown here. For instance, antibodies conjugated to XL665 are supplied on the basis of 20 ng of antibody per well. However, their equivalence in terms of average conjugate quantity per well depends on the molar ratio antibody/XL665, and therefore varies from one toolbox reagent to another. In practice, the active moiety amount is generally preferred to that of total conjugate as a basis for calculating assay development. This is due to the fact that the label moiety does not influence the interaction studied per se. The average conjugate quantity per well is information that reflects overall biological material content. For Eu3+ and Lumi4-Tb cryptates, d2, biotin, and DNP conjugates, the total conjugate amount equals that of the active moiety, since the molecular weight of the label is negligible. Reagents from the HTRF toolbox can be used in multiple configurations. A number of assays can be developed using a simple one-step homogeneous protocol in which interaction occurs simultaneously with detection. The example given here – a study published by Préaudat et al. – describes a two-step protocol for quantifying caspase-3 substrate cleavage (DEVD). Like most HTRF protease systems, the assay is set up using a universal cassette detection system of streptavidin-cryptate and anti-DNPXL665. It is entirely homogeneous and runs in a single plate.

Recommended quantities of conjugates:

Most assays can be run within the nanomolar range. However, as a tracer, cryptate conjugates must not be excessive in order to prevent reader saturation and an unacceptable level of background. In most cases, a Eu3+ or Lumi4-Tb cryptate concentration of 3 to 5 nM is appropriate, and will typically generate 620 nm fluorescence in the range of 20,000 to 80,000 cps depending on the HTRF compatible reader used. As an example, for an antibody conjugated to Eu3+cryptate with a molar ratio of 5 cryptates/Ab, the recommended value would be close to 1 nM of antibody. The XL665 conjugate must match its assay counterpart as closely as possible so that the maximum number of biomolecules can be tagged with the XL665 acceptor. Thus, to detect a GST-tagged molecule at an assay concentration of 20 nM, the concentration of anti-GST-XL665 should be equimolar or higher. The actual amount will depend on the assay configuration and the degree of miniaturization.