TR-FRET and FP Assay Reagents

What is TR-FRET?

TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) is a highly sensitive and widely used assay technology in the field of drug discovery and biological research to study protein-protein interactions, enzyme kinetics, and receptor-ligand binding. This technology works by measuring the energy transfer between two fluorescent molecules - a donor (such as CoraFluor™) and an acceptor. When the donor molecule is excited by a light source, typically 340 nm, energy is transferred from the donor to the acceptor through a phenomenon called Förster resonance energy transfer (FRET) if the two molecules are in proximity (typically 5-10 nm) to each other (Figure 1). TR-FRET assays generally utilize lanthanides as donors, which have a large Stokes shift and millisecond long lifetimes, enabling time-delayed detection and eliminating background noise derived from scattered excitation light and autofluorescence from media components and proteins.

Our TR-FRET toolbox offers a wide range of highly versatile probes to generate biochemical assays.

Figure 1: Schematic outlining the TR-FRET assay principle as applied to induced proximity.

Featured Product: CoraFluor™ Probes

CoraFluor™ 1 (Cat. No. 7920) and CoraFluor™ 2 (Cat. No. 7950) are terbium-based TR-FRET donors that emit wavelengths compatible with commonly used fluorescent acceptor dyes such as FAM or FITC (Cat. No. 5440), BODIPY^® (BDY), Janelia Fluor^® dyes, TMR, and Cyanine 5, SE (Cat. No. 5436), making it easy to incorporate into ongoing screening cascades (Figure 2). Compared to existing TR-FRET donors, the CoraFluor fluorescence is brighter and more stable in biological media, enhancing sensitivity and data generation from biochemical assays. CoraFluor™ 1 exhibits excitation upon exposure to a 337 nm UV laser, whereas CoraFluor™ 2 displays a red-shifted excitation wavelength, enhancing excitation efficiency at 365 nm and 405 nm. This attribute of CoraFluor™ 2 widens the range of analytical instruments suitable for these assays.

CoraFluor reagents can be conjugated directly to your antibody or protein of interest (see protocol). Alternatively, custom services for the conjugation of CoraFluor reagents to an antibody of your choice are available from R&D Systems.

Figure 2: CoraFluor emission spectral overlap with common acceptor dyes.

What is TR-FRET Used For?

TR-FRET has been extensively used to identify novel drug targets, screen compound libraries, and optimize lead compounds in various therapeutic areas, including oncology, cardiovascular disease, infectious disease, and neurodegenerative disorders. TR-FRET assays are particularly useful in measuring the binding affinity of small molecules, such as inhibitors and Degraders, to protein targets in a multi-well plate format, allowing for efficient high-throughput screening of target engagement (Figure 3).

Figure 3: CoraFluor TR-FRET target engagement assay.

TR-FRET in Degrader Development

TR-FRET ternary complex assays are valuable in the development of small molecule protein Degraders as they provide a quantitative measure of the interactions between the Degrader, the target protein, and the ubiquitin E3 ligase (Figure 3). This information is critical for achieving effective targeted protein degradation by characterizing the binding affinity of the Degrader to the target protein, the recruitment of ubiquitin E3 ligase to the target protein, and the formation of a cooperative ternary complex necessary for ubiquitination and subsequent degradation. This allows for optimization of Degrader molecules to improve their potency and selectivity.

Figure 4: CoraFluor TR-FRET ternary complex assay.

It is essential to quantify protein levels accurately. TR-FRET assays present a practical approach for the precise determination of target protein levels in cellular lysates after treatment with a Degrader, leveraging the advantages of a high-throughput format on a fluorescence microplate reader. This technique offers invaluable insights into the time- and dose-dependent kinetics of degradation for a panel of candidate Degraders. Furthermore, it enables calculation of important parameters, including the DC₅₀ (the concentration at which target protein degradation is half-maximal) and D_max (the maximal achievable degradation level) (Figure 5).

Figure 5: CoraFluor TR-FRET protein degradation quantification assay.

What are FP Assays Used For?

Fluorescence polarization (FP) assays are also widely used in biology to study molecular interactions, such as protein-protein, protein-DNA, and protein-ligand binding, as well as for monitoring the progress of enzyme reactions. The technique is based on the observation that the effect of polarized light applied to a fluorescent probe is dependent on multiple factors, such as molecular conformation, orientation, and size. FP assays measure the change in orientation of a molecule in time, between absorption and emission events, and indicate the level of binding of the fluorescent molecule. The approach provides a rapid, quantitative and versatile analysis of molecular interactions, and can be used to measure affinity constants (K_i, K_d) and IC₅₀ values for small molecules. As such FP assays are frequently used for high-throughput screening in drug discovery.

CoraFluor™ is a trademark of Bio-Techne Corp.

BODIPY^® is a registered trademark of Molecular Probes.

Literature for TR-FRET and FP Assay Reagents

Tocris offers the following scientific literature for TR-FRET and FP Assay Reagents to showcase our products. We invite you to request* your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.

Fluorescent Dyes and Probes Research Product Guide

This product guide provides a background to the use of Fluorescent Dyes and Probes, as well as a comprehensive list of our:

• Fluorescent Dyes, including dyes for flow cytometry
• Fluorescent Probes and Stains, including our new MitoBrilliant^TM mitochondria stains
• Tissue Clearing Kits and Reagents
• Aptamer-based RNA Imaging Reagents
• Fluorescent Probes for Imaging Bacteria
• TSA Vivid^TM Fluorophore Kits
• TSA Reagents for Enhancing IHC, ICC & FISH Signals

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TPD and Induced Proximity Research Product GuideUpdated

This brochure highlights the tools and services available from Bio-Techne to support your Targeted Protein Degradation and Induced Proximity research, including:

• Active Degraders
• TAG Degradation Platform
• Degrader Building Blocks
• Assays for Protein Degradation
• Induced Proximity Tools

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