BioAuxilium Thunder™ TR-FRET Cell Signaling Assays
BioAuxilium Thunder™ TR-FRET Cell Signaling Assays

THUNDER, our enhanced TR-FRET technology

THUNDERTM, our enhanced TR-FRET technology

The THUNDERTM TR-FRET assay platform is based on the use of a carefully selected pair of donor-acceptor fluorophores showing exceptional spectral compatibility and TR-FRET signal. THUNDER assays use a long-lifetime and highly stable Europium chelate (Eu) as the donor fluorophore and a small, unique far-red fluorophore (FR) as the acceptor. In a sandwich immunoassay application, one antibody is labeled with the Eu chelate and the second antibody is labeled with the FR fluorophore. The specific binding of the two labeled antibodies to distinct epitopes on the target protein takes place in solution and brings the two fluorophores into close proximity. Excitation of the donor Eu with a flash lamp (320 or 340 nm) or a laser (337 nm) triggers a FRET to the acceptor FR, which in turn emits a specific TR-FRET signal at 665 nm. Residual (non-transferred) energy from the Eu generates light at 615 nm. These long-lived fluorescent signals are read in a time-resolved manner to minimize assay interference, which increases assay performance (Figure 3). In addition, the concentrations of fluorophore-labeled antibodies in all THUNDER assay kits have been carefully optimized to further minimize assay background and maximize the S/B ratio. Of note, the high resistance of the Eu chelate to photobleaching and very long signal stability allows the reading of assays as many times as needed, including the archiving of samples.

Discover THUNDER™ TR-FRET by BioAuxilium The THUNDER™ TR-FRET assay platform is based on the use of a carefully selected pair of donor-acceptor fluorophores showing exceptional spectral compatibility and TR-FRET signal. THUNDER assays use a long-lifetime and highly stable Europium chelate (Eu) as the donor fluorophore and a small, unique far-red fluorophore (FR) as the acceptor. In a sandwich immunoassay application, one antibody is labeled with the Eu chelate and the second antibody is labeled with the FR fluorophore. The specific binding of the two labeled antibodies to distinct epitopes on the target protein takes place in solution and brings the two fluorophores into close proximity. Excitation of the donor Eu with a flash lamp (320 or 340 nm) or a laser (337 nm) triggers a FRET to the acceptor FR, which in turn emits a specific TR-FRET signal at 665 nm. Residual (non-transferred) energy from the Eu generates light at 615 nm. These long-lived fluorescent signals are read in a time-resolved manner to minimize assay interference, which increases assay performance (Figure 3). In addition, the concentrations of fluorophore-labeled antibodies in all THUNDER assay kits have been carefully optimized to further minimize assay background and maximize the S/B ratio. Of note, the high resistance of the Eu chelate to photobleaching and very long signal stability allows the reading of assays as many times as needed, including the archiving of samples.

Figure 3: The binding of Eu-labeled and FR-labeled antibodies to the target protein in the cell lysate brings donor and acceptor molecules into close proximity. Upon excitation of the Europium chelate at 320 or 340 nm, energy is transferred to the far-red acceptor fluorophore, which in turn emits light at 665 nm. The intensity of light emission at 665 nm is proportional to the level of protein.

Another key advantage of TR-FRET is that data can be expressed and analyzed as either the emission signal at 665 nm or the 665 nm/615 nm emission signals ratio. The use of the ratiometric measurement further increases data reproducibility because it normalizes small pipetting errors and also corrects for some compound artifacts. Indeed, some test compounds may quench the donor Eu signal at 615 nm either by absorbing the excitation light at the UV wavelength used to excite the donor (“inner filter effect”), by absorbing the donor emission at 615 nm or by light scattering (e.g., poorly soluble compounds). Because such situations can cause a decrease in the intensity of both the donor and acceptor emission signals, the 665 nm/615 nm ratio corrects for these interferences.


THUNDERTM TR-FRET
cell-based assays present many benefits compared to western blots, ELISA and bead-based approaches.

  • Easy-to-use, fast and homogeneous (no-wash) assays.
  • Low assay background, superior S/B ratios and high sensitivity.
  • High robustness and reproducibility (ratiometric measurements).
  • Very long signal stability.
  • High tolerance to most experimental conditions.
  • Compatibility with most multi-mode microplate readers typically found in research labs.
  • Cost-effectiveness.


Assay Validation Results