UA-Glo® BRET Detection System

The UA-Glo® BRET Assay Kit includes UA-Glo® BRET Ligand 618 and UA-Glo® BRET Micro-Luciferase Substrate. UA-Glo® BRET Ligand 618 can serve as an energy acceptor in NanoBRET® experiments, while UA-Glo® BRET Micro-Luciferase Substrate acts as a substrate for NanoLuc®, generating donor luminescence signals when catalyzed by NanoLuc®. Together, they function as detection reagents for NanoBRET® experiments. Upon entering live cells, UA-Glo® BRET Ligand 618 covalently binds to the Halo-Tag® of fusion protein A (tagged with Halo-Tag®) via its halogen group. When the BRET Ligand 618-labeled Halo-Tag® fusion protein A interacts with fusion protein B (tagged with NanoLuc®) in the presence of a micro-luciferase substrate such as UA-Glo® BRET Micro-Luciferase Substrate, the light energy produced by the reaction between NanoLuc® fusion protein B and the substrate can be absorbed by nearby BRET Ligand 618. The excited BRET Ligand 618 emits red light, which can be quantitatively detected and is proportional to the binding affinity/quantity of proteins A and B. Simultaneously measuring the NanoLuc® luminescence and calculating the ratio of BRET Ligand 618 to NanoLuc® luminescence eliminates experimental errors and background interference, significantly enhancing detection sensitivity.

UA-Glo® BRETDetection Kit components and specifications are as follows:

The NanoBRET assay requires simultaneous measurement of the luminescence intensities of the energy donor NanoLuc® and the energy acceptor BRET Ligand 618. The emission peak of NanoLuc® is at 460 nm, while that of BRET Ligand 618 is at 618 nm. For detecting donor luminescence, a BP-type filter around 460 nm is recommended, such as Em 450nm/BP80. For detecting acceptor luminescence, an LP-type filter around 600-610 nm is recommended, such as Em 610nm/LP. Suitable equipment includes multi-functional microplate readers capable of BRET detection, such as the PerkinElmer EnVision®, BMG Labtech CLARIOstar®, or Promega GloMaxR Discover System. Filter selection and NanoBRET™ detection parameter settings for the microplate reader can be referenced from the equipment manual and relevant literature.

The following experiment demonstrates the application of the UA-Glo® BRET Detection Kit in a NanoBRET® assay, using the example of detecting the effect of compounds on the interaction between Halo-Tag® fusion protein A and NanoLuc® fusion protein B.

1) Co-transfect Halo-Tag® fusion protein A and NanoLuc® fusion protein B into HEK293 cells using transient transfection.

2) Approximately 20 hours post-transfection, collect the cells and resuspend them in phenol red-free Opti-MEM® I medium supplemented with 4% FBS at an appropriate density and volume.

3) UA-Glo® BRET Ligand 618 is provided as a DMSO solution at a concentration of 1000x (0.1 mM). Add it to the cells prepared in step 2) at a final concentration of 1x (0.1 μM) and mix thoroughly. Include a cell control without BRET Ligand 618, adding DMSO to a final concentration of 0.1%.

4) Seed the cells prepared in step 3) into a transparent-bottom white cell plate at an appropriate density: 90 μL per well for a 96-well plate or 36 μL per well for a 384-well plate.

5) Dilute the test compounds to 10x the final test concentration using phenol red-free Opti-MEM® I medium supplemented with 4% FBS, and add them to the cell plate prepared in step 4): 10 μL per well for a 96-well plate (final volume 100 μL) or 4 μL per well for a 384-well plate (final volume 40 μL). Ensure the final DMSO concentration does not exceed 0.5%.

6) Incubate the experimental cell plate at 37°C, 5% CO2 for at least 4-6 hours or overnight (18-24 hours). Overnight incubation is recommended.

7) Prepare the NanoLuc® substrate: Dilute the UA-Glo® BRET micro-luciferase substrate 100-fold in phenol red-free Opti-MEM® I medium.

8) Remove the experimental cell plate and add the substrate prepared in step 7): 25 μL per well for a 96-well plate or 10 μL per well for a 384-well plate. Mix by shaking at medium speed for 30 seconds.

9) Within 10 minutes of adding the substrate, measure the donor (460 nm) and acceptor (618 nm) luminescence signals using a multi-functional microplate reader capable of NanoBRET™ detection.

Data processing: The ratio of acceptor (618 nm) to donor (460 nm) luminescence signals (BU, multiplied by 1000 to obtain mBU) is the NanoBRET™ ratio. Calculate the NanoBRET™ ratio for both samples and the control without BRET Ligand 618. The corrected NanoBRET™ ratio is obtained by subtracting the NanoBRET™ ratio of the control without BRET Ligand 618 from that of the sample.

1) Different batches are not recommended for mixed use 2) Without rigorous validation, it is not advisable to alter the dosage of detection reagents 3) For research use only 4) HaloTag, Nano-Glo, and NanoLuc are registered trademarks of Promega Corporation. NanoBRET is a trademark of Promega Corporation