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Silvia Vidali, PhD | Application Scientist | Molecular Devices

Cathy Olsen, PhD | Sr. Application Scientist | Molecular Devices

Introduction

BRET (bioluminescence resonance energy transfer) is a technique for measuring protein-protein or protein- ligand interactions that involves the interaction of a bioluminescent donor and a fluorescent acceptor. When donor and acceptor are closer than 10 nm to each other, the donor excites the acceptor, which then emits fluorescence. By tagging one protein of interest with the donor and its binding partner with the acceptor, one can measure protein interactions by using a microplate reader to detect light emitted by the donor and acceptor.

NanoBRET™ technology from Promega improves upon earlier generations of BRET, including BRET1 and BRET2, by incorporating a brighter luminescence donor (NanoLuc® luciferase), optimized energy acceptor (HaloTag®-NCT), and a wider separation between donor and acceptor wavelengths (Figure 1). These improvements offer increased signal, better sensitivity, and lower background, enabling detection of protein interactions within the context of living cells¹.

Detection of NanoBRET signals, and analysis of the resulting data, requires sensitive instrumentation and advanced software. The SpectraMax® Multi-Mode Microplate Readers let researchers acquire NanoBRET data using either an optimized filter set or monochromator- based wavelength selection. SoftMax® Pro Software enables scientists to apply analysis with curve fitting to the results. Here, we show two methods we used to validate NanoBRET assay performance on five different microplate readers:

• SpectraMax® iD5 Multi-Mode Microplate Reader
• SpectraMax i3x Multi-Mode Microplate Reader
• SpectraMax Paradigm Multi-Mode Microplate Reader
• SpectraMax M5e Multi-Mode Microplate Reader
• FlexStation® Multi-Mode Microplate Reader

Assay 1: NanoBRET Positive Control

This straightforward method lets you know if your instrument is properly configured to detect the NanoBRET signal. It consists of one vector that tethers together the NanoLuc and HaloTag proteins, ensuring energy transfer with a very strong signal. If you use this method as a positive control in your NanoBRET assay, it is recommended to leave at least one empty well between the Positive Control and your sample, to avoid crosstalk to adjacent wells.

Assay 2: NanoBRET PPI Control Pair (p53/MDM2 interaction)

This method is another way to test proper instrument setup and can also be used as a positive control for your NanoBRET assay. The NanoBRET PPI Control Pair method allows measurement of the interaction of the tumor suppressor p53 and the oncoprotein MDM2. The p53 pathway activator nutlin-3 is used to disrupt the p53-MDM2 interaction in a concentration-dependent manner, with results graphed as a dose-response curve.

Materials and methods

Reagents

• NanoBRET Positive Control (Promega cat. #N1581), including:
  • NanoBRET Positive Control Vector (1 μg/μL)
  • Transfection Carrier DNA (1 μg/μL)
• NanoBRET PPI Control Pair
• (p53, MDM2; Promega cat. #N1641)
• NanoBRET Nano-Glo® Detection System (Promega cat. #N1661)
• Nutlin-3 (Sigma-Aldrich cat. #SML0580)
• HEK293 cells (ATCC cat. #CRL-1573)
• MEM (Minimum Essential Medium, ThermoFisher Scientific cat. #11095080)
• Penicillin-Streptomycin (10,000 U/mL, ThermoFisher Scientific cat. #15140122)
• Fetal bovine serum
• (e.g., VWR cat. #97068-085)
• Opti-MEM Reduced-Serum Medium (ThermoFisher Scientific cat. #11058-021)
• FuGENE® HD or FuGENE 4K Transfection Reagent (Promega cat. #E2311 or #E5911)
• 6-well clear microplate (Costar cat. #3506)
• Solid white 96-well plate, tissue culture treated (Corning cat. #3917)

Figure 1. How a NanoBRET assay works. When a NanoLuc-Protein A fusion (energy donor) interacts with a fluorescently labeled HaloTag-Protein B fusion (energy acceptor), donor and acceptor are brought close together and energy is transferred.

Microplate readers

• SpectraMax M5e Multi-Mode Microplate Reader*
• FlexStation 3 Multi-Mode Microplate Reader*
• SpectraMax iD5 with the following emission filters:
  • 447 nm (Molecular Devices P/N 6590-0088)
  • 610 nm (Molecular Devices P/N 6590-0117)
• SpectraMax i3x Multi-Mode Microplate Reader with NanoBRET Detection Cartridge
• SpectraMax Paradigm Multi-Mode Microplate Reader with NanoBRET Detection Cartridge

*SpectraMax M5e and FlexStation 3 readers contain equivalent hardware for luminescence detection and will yield comparable NanoBRET results.

Methods

Assay 1: NanoBRET Positive Control

HEK-293 cells were trypsinized and diluted in culture medium (MEM + 10% fetal bovine serum + 1% penicillin/ streptomycin) to 400,000 cells per mL. 2 mL of cells (800,000 cells) were placed in each well of a 6-well plate. The cell plate was then incubated for 5 hours at 37°C and 5% CO2.

Transfection mixtures containing Positive Control Vector or carrier only were made in two sterile microcentrifuge tubes. In one tube, 0.002 μg of NanoBRET Positive Control Vector was combined with 2 μg of Transfection Carrier DNA. To the other tube, 2 μg of Transfection Carrier DNA was added. 100 μL of Opti-MEM Reduced-Serum Medium was added to each tube, and contents were mixed. 8 μL of FuGENE 4K Transfection Reagent was added to each tube, and mixtures were incubated at room temperature for 10 minutes. Transfection mixtures were added dropwise to two of the wells of the 6-well cell plate, and carrier-only mix was added to one well. Cells were incubated for 20 hours at 37°C and 5% CO2.

The next day, transfection media were aspirated, and transfected cells were rinsed 1X with 1 mL PBS per well. Cells were trypsinized and resuspended in 2 mLs of culture medium per well, then counted. Cells were spun down, supernatant was discarded, and cells were resuspended in Opti-MEM + 4% FBS to a final density of 2 x 105 cells/mL. To half of each sample, 1 μL of 0.1 mM HaloTag NanoBRET 618 Ligand per mL of cells was added (100 nM final concentration), and to the other half DMSO (0.1% final concentration was added as a no-ligand control. For each cell suspension, 100 μL was dispensed into eight wells of a solid white 96-well microplate, followed by incubation for 20 hours at 37°C and 5% CO2.

Following incubation, 25 μL of a 5X solution of NanoBRET Nano-Glo Substrate in Opti-MEM (100-fold dilution of stock) was added to each assay well. The microplate was mixed for 30 seconds on a titer plate shaker and read on the SpectraMax readers, using the settings shown in Table 1.

*Read height can be optimized for the microplate used.
Table 1. NanoBRET detection settings. SpectraMax i3x and Paradigm readers must be equipped with a NanoBRET detection cartridge, and the SpectraMax iD5 reader requires the two emission filters indicated in Materials, above.

Assay 2: NanoBRET PPI Control Pair (p53/MDM2 interaction)

HEK-293 cells were resuspended in cell culture medium at 400,000 cells per mL. Two mL were plated per well (800,000 cells/well). Cells were allowed to attach to the wells for 4 to 6 hours at 37°C, 5% CO2.

A transfection mixture was prepared by adding 2 μg p53- HaloTag® Fusion Vector DNA and 0.2 μg NanoLuc®-MDM2 Fusion Vector DNA to 100 μL Opti-MEM Reduced-Serum Medium. Subsequently, 6 µL of FuGENE HD Transfection Reagent was added, and the mix was incubated at room temperature for 10 minutes. The mixture was added to the attached cells, which were incubated for 20 to 24 hours at 37°C, 5% CO2 to allow transfection and protein expression to occur.

Transfected HEK-293 cells were collected by spinning down at 1,000 rpm for five minutes, and the culture medium was discarded. Cell density was adjusted to 2.2 x 105 cells per mL in Opti-MEM + 4% FBS and divided into two 15-mL conical tubes. One tube was treated with 1 µL of 0.1 mM HaloTag 618 Ligand per mL of cell suspension, and the other was treated without ligand (1 µL DMSO per mL of cell suspension). The cells were plated into a 96-well white microplate at 90 µL per well. Cells were immediately treated with either a serial dilution of nutlin-3 (n = 4 replicates per concentration) or 0.5% DMSO. The cells were incubated overnight at 37°C, 5% CO₂.

A 5X solution of NanoBRET Nano-Glo substrate was made in Opti-MEM, and 25 μL were added to each well. Donor emission (447 nm) and acceptor emission (610 nm) were measured on the aforementioned microplate readers using the settings shown in Table 1.

Data analysis (both assays)

NanoBRET ratios were calculated in SoftMax Pro Software by dividing acceptor signal by donor signal, and ratios were multiplied by 1000 to obtain milliBRET units (mBU). For the Positive Control Vector results, corrected NanoBRET ratios were calculated by subtracting mBU values for samples receiving no ligand from those of ligand-treated samples. Z’ factors were also calculated from the results for each instrument (n = 8).

For the PPI Control Pair results, the mBU values were background corrected by subtracting mean mBU for samples containing no ligand. Results for nutlin-3-treated cells were analyzed and graphed using a 4-parameter curve fit in SoftMax Pro Software (version 7.0.3 and higher). Z’ factors were calculated at each concentration of nutlin-3 to assess assay performance.

Results

NanoBRET Positive Control results for the SpectraMax iD5 reader are shown in Figure 2. Table 2 presents summarized results for the four SpectraMax readers featured here. Corrected NanoBRET ratios ranged from 188.0 mBU to 783.3, but the Z’ factors calculated from these results were from 0.92 to 0.99 for all of the SpectraMax readers.

For the NanoBRET PPI Control Pair, NanoBRET ratio (mBU) vs. nutlin-3 concentration was graphed using a 4-parameter curve fit in SoftMax Pro Software (Figure 3). The calculated IC₅₀ value for nutlin-3 was between 1.02 and 1.31 μM, similar to results shown by Promega for the NanoBRET PPI Control Pair².

For FlexStation 3 and SpectraMax iD5, Z’ factors of at least 0.7 were obtained for concentrations of nutlin-3 of 1 µM and below. For SpectraMax i3x and SpectraMax Paradigm, Z’ factors above 0.8 were obtained for all concentrations of nutlin-3. For concentrations of 1 μM and lower, Z’ factors were equal to or greater than 0.9. These values demonstrate the robustness and low variability of this NanoBRET assay, especially for SpectraMax i3x and SpectraMax Paradigm microplate readers.

Figure 2. NanoBRET ratio and raw donor and acceptor measurements with NanoBRET Positive Control vector. Results shown are for the SpectraMax iD5 reader, and similar results were obtained with the other SpectraMax readers tested. A, NanoBRET ratio (in milliBRET units) for experimental sample and no-ligand control. B, Raw donor RLU (relative light units). C, Raw acceptor RLU, representing donor-to-acceptor energy transfer.

Table 2. Luminescence values measured for the NanoBRET Positive Control vector assay. Donor and acceptor emission (RLU), NanoBRET ratios (mBU), and corrected NanoBRET ratios are shown for the SpectraMax readers tested. Results shown are from the same assay plate, with measurements taken on all four instruments within ten minutes.

Figure 3. Disruption of p53-MDM2 interaction by nutlin-3. Results are plotted using a 4-parameter curve fit in SoftMax Pro Software with the formula y = D + (A-D)/[1+(x/C)B], (n = 3 or 4). A, SpectraMax iD5; B, SpectraMax i3x; C, SpectraMax Paradigm; D, FlexStation 3.

Conclusion

The SpectraMax and FlexStation readers featured here, each with settings optimized for detection of NanoBRET donor and acceptor signals, showed excellent results for both the Positive Control Vector and NanoBRET PPI Control Pair (p53, MDM2) assays. Z’ factors for the Positive Control vector of 0.92 to 0.99 validate the performance of the assay on these microplate readers. Disruption of p53-MDM2 interaction in a concentration-dependent manner by nutlin-3 yielded highly reproducible IC₅₀ values, ranging from 1.02 to 1.31 μM on the different instruments. Z’ factors of 0.7 and higher confirm the robust performance of both the microplate readers and the NanoBRET PPI Control Pair assay. SoftMax Pro Software was configured to calculate NanoBRET ratios and graph the data automatically, streamlining the analyses.

References

1. Machleidt T, Woodroofe CC, Schwinn MK, Mendez J, Robers MB, Zimmerman K, Otto P, Daniels DL, Kirkland TA, and Wood KV. NanoBRET—A Novel BRET Platform for the Analysis of Protein-Protein Interactions. ACS Chem. Biol. 2015, 10, 1797–1804.
2. Technical Manual: NanoBRET Protein: Protein Interaction System. Promega Corporation.

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