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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.
TR-FRET exploits the unique photophysical properties of lanthanide labels when used as FRET donors (Figure 2). Lanthanide labels have exceptionally long excited state lifetimes ranging from hundreds of microseconds to a few milliseconds. This is in sharp contrast to the very short fluorescent lifetime, on the low nanosecond range, of both conventional fluorophores used in standard FRET assays and natural background fluorescence. The long fluorescence decay after excitation allows time-resolved or gated FRET signal detection, after all interfering short-lived signals have decayed to negligible levels. The time-resolved detection also avoids interference from the acceptor emission due to its direct excitation (i.e., non-sensitized emission), rather than by FRET. In addition, lanthanide labels exhibit a large Stokes shift (the difference between the excitation and emission maxima) that minimizes crosstalk. Overall, because of their excellent temporal and spectral resolution, TR-FRET assays exhibit very low background and high S/B ratios, two features critical for the development of reproducible and robust assays. Of note, many microplate readers supporting TR-FRET measurements are available
SZABO-SCANDIC offers TR-FRET compatible readers from BioTek.
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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 corrects 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.
Link zum Video: Discover Thunder™ FRET
https://www.youtube.com/watch?time_continue=23&v=E4MYNzUCh2g&feature=emb_logo
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