Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) is a preferred fluorescent assay formats in drug discovery laboratories. TR-FRET assays are less susceptible to compound interference than other assay formats and may be applied to multiple target classes. To support this technology focus, Invitrogen has developed LanthaScreen kinase activity assays. Invitrogen uses two sets of FRET-pairs in developing our LanthaScreen Assays: Terbium/Fluorescein (or GFP) and Europium/AlexaFluor 647.
The LanthaScreen kinase activity assay format is based on the use of a long-lifetime terbium or europium chelate as the donor species and fluorescein as the acceptor species. When terbium (or europium) and fluorescein (or AlexaFluor 647) labeled molecules are brought into proximity, energy transfer takes place causing an increase in acceptor fluorescence and a decrease in donor fluorescence. These fluorescent signals can be read in a time-resolved manner to reduce assay interference and increase data quality.
The time-resolved spectra above illustrate energy transfer occurring when terbium and fluorescein are brought into proximity via biomolecular interactions. The TR-FRET value is determined as a ratio of the FRET-specific signal measured with a 520 nm filter to that of the signal measured with a 495 nm filter, which is specific to terbium. The inset shows the time-resolved spectra in the absence of energy transfer.
Overcome compound interference using LanthaScreen Assays
TR-FRET assays offer advantages over fluorescent polarization (FP) assays when background fluorescence is a problem. In FP assays, background fluorescence due to fluorescent library compounds is often depolarized. Background signal due to scattered light, like that from precipitated compounds, is often polarized. Either phenomenon can lead to a false positive or false negative result, depending on the assay configuration. Because the donor species used in a TR-FRET assay has a fluorescent lifetime that is many orders of magnitude longer than background fluorescence or scattered light, energy transfer can be measured after the interfering signal has completely decayed. Additionally, unlike FP assays, TR-FRET assays can be formatted using limiting receptor and excess tracer concentrations, driving potential cost savings. Our LanthaScreen assays are resistant to interference from color quenchers, light scatterants, and fluorescent compounds.
LanthaScreen Tb exploits Terbium as the donor chemistry
Many TR-FRET assays use europium as the ‘long lifetime label’ doner paired with various far-red acceptors (including allophycocyanin (APC)). Due to the large molecular mass of APC (>100 KD) it has typically been used as a streptavidin conjugate, to in-directly couple to the biotinylated substrate in a trimolecular FRET complex. In contrast, LanthaScreen Tb utilizes terbium as the long lifetime label, enabling direct coupling to fluorescein as the acceptor species. This has the immediate advantage of being able to overcome several issues common to use of APC as the acceptor:
- Simplify assay optimization. Since the donor-acceptor pair in a terbium-based TR-FRET assay does not require a biotin-avidin mediated interaction, three-compound matricies are not required to determine optimal reagent concentrations.
- Avoid problems due to steric bulk associated with the large streptavidin-APC reagents including long incubation times to reach equilibrium.
- Reduce assay cost by replacing streptavidin-APC with fluorescein (as well as remove lot-to-lot variations in streptavidin-APC).
Use of LanthaScreen Tb also enables the use of GFP as the acceptor molecule, which allows for:
- Development of protein substrates expressed as GFP-fusion proteins.
- Development of cell lines expressing GFP-fusions for detection of phosphorylation which has occurred in a live-cell context via TR-FRET.
If a traditional Europium/Far Red FRET pair is preferred, Invitrogen offers all of our peptide-based substrates and matched antibodies with this alternative dye pair. Please contact us at firstname.lastname@example.org for more information.
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