UA-Glo® BRET Nano Luciferase Substrate

The UA-Glo® BRET MicroLuciferase Substrate can serve as a substrate for NanoLuc® in live-cell NanoBRET® protein interaction assays, generating donor luminescence signals upon reaction with NanoLuc®. Meanwhile, the UA-Glo® BRET Ligand 618 acts as an energy acceptor in NanoBRET® assays. When used together, these reagents enable NanoBRET® detection. The UA-Glo® BRET Ligand 618 can enter live cells and covalently bind to the Halo-Tag® of fusion protein A via its halogen group. When the BRET Ligand 618-labeled Halo-Tag® fusion protein A interacts with NanoLuc®-tagged fusion protein B in the presence of a NanoLuc® substrate (e.g., UA-Glo® BRET MicroLuciferase Substrate), the light energy produced by the NanoLuc® fusion protein B and its substrate can be absorbed by nearby BRET Ligand 618. Upon excitation, the BRET Ligand 618 emits red light, which can be quantitatively detected and is proportional to the binding affinity/quantity of proteins A and B. Additionally, measuring the NanoLuc® luminescence and calculating the ratio of BRET Ligand 618 to NanoLuc® signals helps eliminate experimental errors and background interference, significantly enhancing detection sensitivity.

UA-Glo® BRET Micro Luciferase Substrate Specifications are as follows:

NanoBRETTM detection requires simultaneous measurement of luminescence intensity from both 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 emission, a BP-type filter around 460 nm is recommended, such as Em 450 nm/BP80. For detecting acceptor emission, an LP-type filter near 600-610 nm is recommended, such as Em 610 nm/LP. Suitable equipment includes multifunctional plate readers capable of BRET detection, such as PerkinElmer EnVision®, BMG Labtech CLARIOstar®, and Promega GloMaxR Discover System. Filter selection and parameter settings for NanoBRETTM detection can be referenced from equipment manuals and relevant literature.

The following experiment illustrates the application of UA-Glo® BRET micro-luciferase substrate in NanoBRET® assays, using the interaction between Halo-Tag® fusion protein A and NanoLuc® fusion protein B as an example.

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

2)Approximately 20 hours post-transfection, harvest 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 (Youbio, L9001) is provided as a DMSO solution at 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 control without BRET Ligand 618, adding DMSO to a final concentration of 0.1%.

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

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

6)Incubate the 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 UA-Glo® BRET micro-luciferase substrate 100-fold in phenol red-free Opti-MEM® I medium.

8)Remove the cell plate and add the substrate prepared in step 7): 25 μL per well for 96-well plates or 10 μL per well for 384-well plates. 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 multifunctional plate reader capable of NanoBRETTM detection.

10)Data processing: The ratio of acceptor (618 nm) to donor (460 nm) signals (BU; multiplying this ratio by 1000 yields mBU) represents the NanoBRETTM ratio. Calculate the NanoBRETTM ratio for both samples and the no-BRET Ligand 618 control. The corrected NanoBRETTM ratio is obtained by subtracting the control ratio from the sample ratio.

1) Mixing different batches is not recommended

2) Do not alter the amount of detection reagent without rigorous validation

3) For research use only

4) HaloTag, Nano-Glo, and NanoLuc are registered trademarks of Promega Corporation. NanoBRET is a trademark of Promega Corporation