The recruitment of Gαi/Gαs to GPR84 was measured using the Promega NanoBiT Protein-Protein Interaction System. In brief, HEK293 cells seeded at 5 × 104 cells/well on 96-well plates were co-transfected with plasmids encoding GPR84-LgBiT and SmBiT-mini Gαi or Gαs at the ratio of 1:1. Twenty-four hours later, cells were replaced with 40 μl of fresh culture medium (without FBS). And then added 10 μl Nano-Glo Live Cell reagent according to the manufacturer’s protocol (Promega, Cat No: N2011), incubated in a 37 °C, 5% CO2 incubator for 10 min. Another 25 μl culture medium with various concentrations of compounds were added to the cells. After incubation at room temperature for 10 minutes, bioluminescence was measured with an EnVision multiplate reader (PerkinElmer). All functional data are processed in GraphPad Prism 8.0.
Nanobit protein protein interaction system
Manufactured by Promega
The NanoBiT Protein:Protein Interaction system is a bioluminescent reporter assay that enables the detection and measurement of protein-protein interactions in live cells. It uses two small subunits of NanoLuc luciferase that associate upon binding of the proteins of interest, generating a quantifiable luminescent signal.
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Lab products found in correlation
Envision multiplate reader, by PerkinElmer (1 mentions)
Nano glo live cell reagent, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
Synergy h1 hybrid plate reader, by Agilent Technologies (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Furimazine, by Promega (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Glomax 96 microplate luminometer, by Promega (1 mentions)
Glomax detection system, by Promega (1 mentions)
4 protocols using nanobit protein protein interaction system
1
Measuring Gα Recruitment to GPR84 via NanoBiT Assay
2
NanoBiT Assay for RCAN1-CaN Interaction
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The NanoBiT Protein:Protein Interaction system (Promega, #N2014) was used for the binding assay of RCAN1-CaN interaction according to the manufacturer’s protocol. HEK293 cells plated in a 96-well plate were transfected with 25 ng (per well) of pBiT1.1-N-RCAN1 (89-197) and pBiT2.1-C-CaN (1-391) using PEI (Polysciences, 24765) with Opti-MEM (Life Technologies, 31985). Forty-eight hours after transfection, 25 μL of Nano-Glo live Cell Assay reagent was added to each well after autophagy inducers were treated. After the initial measurement, luminescence values were measured every 30 minutes using the Synergy H1 Hybrid plate reader (BioTek).
3
NanoBiT Protein Interaction Assay
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PCA was performed using NanoBiT® Protein:Protein Interaction System (Promega). Twenty-four hours after transfection of 293T cells in a six-well plate with the indicated genes in the NanoBiT vectors, cells were washed in PBS and resuspended in 1 ml of Opti-MEM™ I reduced serum medium (Thermo Fisher Scientific), and 100 µl of the cell suspension was transferred to a 96-well white plate in triplicate. Furimazine (N1110, Promega), a NanoLuc®-luciferase substrate, was diluted in PBS at a ratio of 1 to 50 and 25 µl of the diluted substrate was added to each well. After 5 min of incubation, the luciferase activity in each well was measured by GloMax® 96 microplate luminometer (Promega).
4
High-throughput Screening of HBx-DDB1 Inhibitors
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The NanoBiT Protein:Protein Interaction system (Promega) was used to establish a screening assay for inhibition of the HBx– DDB1 interaction. Briefly, we transiently transfected 1 μg of HBx and DDB1 with split luciferases into HEK293T or HepG2 cells, at 80% confluence in a 10-cm dish. After 10 hours, cells were reseeded onto a 96-well plate with 5 × 104 cells/well and 50 μL medium/well. After 10 hours of incubation at 37°C, 12.5 μL of Nano-Glo live Cell Assay reagent was added to each well, and baseline luminescence was measured using a luminometer. Immediately after this initial measurement, compounds were added to a final concentration of 10 μM, and luminescence values were measured every 30 minutes using the GloMax Detection System (Promega). To calculate Z’-scores, used as a screening quality indicator, blank wells containing only media were set up with luciferase assay reagents in the first and last columns to determine the Average 0 and standard deviation (SD) 0. Then, transfected cells were seeded into all other columns with media, and DMSO was added followed by luciferase reagents to determine the Average DMSO and SD DMSO. The Z’ score was defined as 1 − ((3 × SD DMSO) + (3 × SD 0)/ (Average DMSO – Average 0)), and a screening system was considered acceptable if it was over 0.5.31 (link)
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