UA-Glo®BRET Intracellular TE Substate/Inhibitor

UA-Glo® BRET Intracellular TE Substrates and Inhibitors include Youbio Biotech's proprietary micro-luciferase substrates and micro-luciferase inhibitors, which can be used to detect the binding (Target Engagement, TE) of micro-luciferase (NanoLuc®, NLuc) fused target proteins with compounds in cells. In the NanoBRET® TE experimental design, the NLuc-fused target protein serves as the energy donor for NanoBRET®, while the target protein-specific binding tracer acts as the energy acceptor. The light energy generated by the NLuc-catalyzed reaction of the micro-luciferase substrate from the NLuc-fused target protein can be absorbed by the tracer bound to the target protein nearby, and the light emitted by the excited tracer can be quantitatively detected. When a test compound binds to the target protein and competes with the tracer, the amount of tracer bound to the target protein decreases, resulting in a corresponding reduction in the emitted light energy. The strength of the tracer's emitted light can be quantitatively measured to calculate the degree of intracellular binding between the test compound and the target protein. Simultaneously detecting the NLuc luminescence value and calculating the ratio of the tracer to NLuc luminescence values can eliminate experimental errors and background interference, significantly improving detection sensitivity. The micro-luciferase inhibitor, added alongside the micro-luciferase substrate during detection, cannot enter cells but can inhibit the micro-luciferase activity of any extracellular NLuc-fused target proteins, further enhancing detection sensitivity.

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NanoBRET® experiments require simultaneous detection of the luminescence intensity of the energy donor NanoLuc® and the energy acceptor. The peak emission wavelength of NanoLuc® is 460nm, while the chromophore of the tracer (NanoBRET® 590 Dye or 618 Dye) emits at 590-610nm. For detecting donor luminescence, a BP-type filter near 460nm (bandwidth 8–80nm) is recommended, such as Em 450nm/BP80. For acceptor luminescence, an LP-type filter near 600-610nm is suggested, such as Em 610nm/LP. Suitable equipment includes multi-functional plate readers capable of BRET detection, such as PerkinElmer EnVision®, BMG Labtech CLARIOstar®, and Promega GloMaxR Discover System. Filter selection and instrument settings for NanoBRET® detection can be referenced from device manuals and relevant literature.

The following experiment demonstrates the application of UA-Glo® BRET intracellular TE substrates and inhibitors in NanoBRET® TE assays, using the measurement of compound EC₅₀ for NLuc-fused target proteins as an example. Detailed HEK293 transfection methods, compound and tracer usage can be referenced from relevant literature and product manuals, such as Promega's Intracellular TE Nano-Glo® Substrate/Inhibitor, Intracellular TE Nano-Glo® Vivazine™/Inhibitor, and NanoBRET® Target Engagement Intracellular Kinase Assay, Adherent Format, or optimized by users based on specific experiments.

1)Transiently transfect HEK293 cells with plasmids encoding NLuc-fused target proteins.

2)Seed transfected cells at an appropriate density in a 96-well clear-bottom white cell culture plate (100μL/well). The recommended seeding medium is Opti-MEM® I low-serum medium (phenol red-free) +1% FBS.

3)Incubate the cell plate at 37℃, 5% CO₂ overnight (20-30hr).

4)Prepare 20x tracer: After 20-30hr incubation, prepare the tracer at 20x final concentration according to literature/product manual recommendations or user optimization. First dilute the tracer in DMSO to 100x, then further dilute to 20x (5% DMSO) using tracer dilution buffer. Use polypropylene tubes to minimize wall adsorption.

5)Prepare test compounds: Serially dilute compounds in DMSO to 1000x final concentration, then dilute to 10x in Opti-MEM® I low-serum medium (phenol red-free).

6)Add 5μL 20x tracer and 10μL 10x test compound to each well. Include controls (solvent control, positive compound control, tracer-free solvent control, etc.). Mix by shaking for 15sec.

7)Incubate at 37℃, 5% CO₂ for 2hr or user-optimized duration.

8)Prepare 3x intracellular TE detection reagent: Dilute the micro-luciferase substrate to 167x and the micro-luciferase inhibitor to 500x in Opti-MEM® I low-serum medium (phenol red-free). Mix gently by inverting 5-10 times. Prepare fresh and discard unused reagent.

9)Remove the cell plate and equilibrate to room temperature.

10)Add 50μL 3x intracellular TE detection reagent to each well. Incubate at room temperature for 2min.

11)Measure donor (e.g., 460nm) and acceptor (e.g., 610nm) luminescence signals using a NanoBRET^TM-compatible multi-functional plate reader. Read within 10min after adding detection reagent.

12)Data processing: The ratio of acceptor (610nm) to donor (460nm) signals (BU, multiplied by 1000 for mBU) represents the NanoBRET^TM ratio. Calculate the corrected NanoBRET^TM ratio by subtracting the tracer-free control ratio from the sample ratio.

1) Different batches are not recommended for mixed use 2) Do not alter the amount of detection reagents without rigorous validation 3) For research use only 4) NanoLuc and NanoBRET are registered trademarks of Promega Corporation