UA-Glo® BRET Ligand 618

UA-Glo® BRET Ligand 618 is a red-emitting fluorescent dye after excitation, which can serve as an energy acceptor in BRET (Bioluminescence Resonance Energy Transfer) experiments, particularly suitable for detecting protein-protein interactions in live cells in NanoBRET™ assays. UA-Glo® BRET Ligand 618 can enter live cells and covalently bind to Halo-Tag® fusion protein A via its halogen group and the Halo-Tag®. When the BRET Ligand 618-labeled Halo-Tag® fusion protein A interacts with NanoLuc® fusion protein B, and in the presence of a NanoLuc® substrate such as UA-Glo® BRET Micro Luciferase Substrate, the luminescence produced by the reaction between NanoLuc® fusion protein B and the substrate can be absorbed by the nearby BRET Ligand 618. The red light emitted by the excited BRET Ligand 618 can be quantitatively detected and is directly proportional to the extent/amount of protein A and B interaction. Simultaneously measuring the NanoLuc® luminescence and calculating the ratio of BRET Ligand 618 to NanoLuc® luminescence can eliminate experimental errors and background interference, significantly enhancing detection sensitivity.

UA-Glo® BRET Ligand 618 specifications are as follows:

NanoBRETTM detection requires simultaneous measurement of the luminescence intensity of both the energy donor NanoLuc® and the energy acceptor BRET Ligand 618. The emission peak of NanoLuc® is at 460nm, while that of BRET Ligand 618 is at 618nm. For detecting donor luminescence, a BP filter around 460nm is recommended, such as Em 450nm/BP80. For detecting acceptor luminescence, an LP filter around 600-610nm is recommended, such as Em 610nm/LP. Suitable equipment includes multi-mode plate readers capable of BRET detection, such as the PerkinElmer EnVision®, BMG Labtech CLARIOstar®, and Promega GloMaxR Discover System. Filter selection and NanoBRETTM detection parameters can be referenced from the equipment manuals and related literature.

The following experiment demonstrates the application of UA-Glo® BRET Ligand 618 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 using transient transfection.

2)After approximately 20 hours of 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 is provided as a DMSO solution at 1000x (0.1mM). Add it to the cells prepared in step 2) at a final concentration of 1x (0.1μM) 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) into a clear-bottom white cell plate at an appropriate density: 96-well plate (90μL per well) or 384-well plate (36μL per well).

5)Dilute the test compounds in phenol red-free Opti-MEM® I medium with 4% FBS to 10x the final test concentration. Add to the cell plate prepared in step 4): 10μL per well for 96-well plates (final volume 100μL) or 4μL per well for 384-well plates (final volume 40μL). 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 the UA-Glo® BRET micro-luciferase substrate (Youbio, L9002) 100-fold in phenol red-free Opti-MEM® I medium.

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

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

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