Nanobret nano glo substrate

NanoBRET assay was performed according to the manufacturer’s instructions. Briefly, 8 × 10₅ of Huh7 cells were seeded onto a 6-well plate and incubated for 6 h at 37°C, 5% CO₂. The cells were transfected with indicated concentrations of pHaloTaq-tagged NS5A and pNLF1-tagged IKKε plasmids (Promega). Each DNA was mixed with Opti-MEM I Reduced Serum Medium, no phenol red (Gibco, USA), and lipofectamine 2000 transfection reagent and incubated for 10 min at room temperature. Cells were transfected with DNA mixture and incubated 20 h at 37°C, 5% CO₂. Cells were washed with Dulbecco’s phosphate buffered saline (DPBS), resuspended with trypsin neutralization solution and then centrifuged at 125 × g for 5 min. Cells were resuspended in an equal volume of assay medium (Opti-MEM I Reduced Serum Medium, no phenol red + 4% FBS). Approximately 2 × 10₅/ml cells were seeded onto a 96-well plate and incubated for 18 h at 37°C, 5% CO₂. Twenty-five microliters of NanoBRET Nano-Glo substrate (Promega) was added into cells and plate was shaken for 30 s. Mean NanoBRET ratio value was determined by measuring the donor emission (460 nm) and acceptor emission (618 nm).

Twenty-four hours after the transient transfection of the NanoLuc and HaloTag constructs, the cells were washed with PBS and trypsinized. Cells were counted and diluted in OptiMEM supplemented with 4% fetal bovine serum to reach a density of 2 × 10⁵ cells/ml. One microliter of 0.1 mM HaloTag NanoBRET ligand 618 (Promega) per ml of cells, or DMSO in the control conditions, was added. One hundred microliters of cells with either ligand or DMSO were plated in a 96-well plate (Corning 3917, Thermo Fisher Scientific) and were cultured overnight at 37 ˚C, 5% CO₂. Twenty-five microliters of 100× dilution of NanoBRET NanoGlo substrate (Promega) was added per well. Plates were shaken for 30 s at 400 rpm and then kept in the dark for 30 s. Finally, donor emission (447 nm) and acceptor emission (610 nm) were measured at room temperature using a dual filter (SpectraMax i3x, Molecular Devices). For all experiments, biological triplicates were performed. All biological triplicates were composed of technical triplicates.
Relative light units obtained for donor and acceptor emission were processed as follows to obtain milli bioluminescence units: $\frac{emission610\text{nm}}{emission447\text{nm}}=BU\times 1000=mBU$
For saturation experiments, percentages of the maximum NanoBRET ratio were plotted against the acceptor to donor ratio; data were fitted using the “one site–total and nonspecific binding” fit in Prism.