pHA-D2LR was purchased from UMR cDNA Resource Center (www.cdna.org ). The plasmid pMOR-RLuc was a kind gift from Dr. Francisco Ciruela (University of Barcelona, Barcelona, Spain), pD2LR-mCherry was a kind gift from Dr. Ibeth Guevara-Lora (Jagiellonian University, Krakow, Poland), and pmCherry-CAAX was from Dr. Deepak Saini’s laboratory (Indian Institute of Science, Bangalore, India). The plasmids pMOR-YFP, pFLAG-D2SR, pD2LR-YFP, pD2LR-RLuc, and pEYFP were kindly provided by Dr. Kjell Fuxe (Karolinska Institutet, Stockholm, Sweden). The plasmid pEGFP was procured from Clontech Laboratories (Saint-Germain-en-Laye, France). The sequences encoding human D2LR and MOR were amplified using the primers described in Table S1 using an MJ Research PTC-200 Thermal Cycler (GMI, MN, USA) and cloned in the NanoBiT® system from Promega (Madison, WI, USA). Using this system, the split fragments of nanoluciferase, namely LargeBiT (LgBiT, an 18kD protein) and SmallBiT (SmBiT, an 1kD peptide) were fused C-terminally to the D2LR and MOR. HaloTag-SmBiT was also procured from Promega. D2LR-EGFP was constructed by digestion of D2LR-LgBiT with HindIII and EcoRI and subcloning into the pEGFP-N3 vector (Addgene, Watertown, MA, USA). The constructs containing the appropriate inserts were verified by restriction digest and by sequencing.
Nanobit system
Manufactured by Promega
Sourced in United States
The NanoBiT® system is a protein-protein interaction detection tool that uses a split NanoLuc® Luciferase technology. It allows for the study of protein-protein interactions in living cells and cell-free systems.
Automatically generated - may contain errors
Lab products found in correlation
Pegfp n3 vector, by Addgene (1 mentions)
Baculovirus method, by Expression Systems (1 mentions)
Coelenterazine, by Yeasen (1 mentions)
Nano glo live cell substrate, by Promega (1 mentions)
Hepes buffered f12 dmem, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Pherastar microplate reader, by BMG Labtech (1 mentions)
4 protocols using nanobit system
1
Bioluminescent Receptor Constructs
2
GPCR-G Protein Interaction Assay
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Analysis of Gi-protein recruitment was performed by using a modified protocol of the NanoBiT system (Promega) described previously30 . NanoBiT system is a two-subunit system based on NanoLuc luciferase that can be used for functional detection for binding and dissociation of GPCR and G-protein. LgBiT (17.6 kDa) of the NanoBiT luciferase was fused to the C-terminal of GPCR with 15-amino acid flexible linkers. SmBiT was C-terminally fused to Gβ subunit with a 15-amino acid flexible linker. Human 5-HT1A-LgBiT, 5-HT1D-LgBiT, and 5-HT1e-LgBiT, WTGαi1, SmBiT-fused Gβ1, and Gγ2 were co-expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus method (Expression Systems). Cell cultures were grown to a density of 2-3 million cells per ml and then infected with four separate baculoviruses at a suitable ratio. The culture was collected by centrifugation 48 h after infection and cell pellets were collected with PBS. The cell suspension was dispensed in a white 384-well plate at a volume of 40 μl per well and loaded with 5 μl of 90 μM coelenterazine (Yeasen) diluted in the assay buffer. Test compounds (5 μl) were added and incubated for 3-5 min at room temperature before measurement. Luminescence counts were normalized to the initial count and fold-change signals over vehicle treatment were used to show G-protein binding response.
3
Nano-Luciferase Protein Interaction Assay
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The NanoBiT® system from Promega was employed, using fusion constructs of MOR and Nb39 with split fragments of a modified nanoluciferase (NanoLuc). The generation of MOR-NanoBiT® fusion constructs has been described previously [16] (link). The different Nb39-NanoBiT ® fusion constructs (Nb39-LgBiT, LgBiT-Nb39, Nb39-SmBiT and SmBiT-Nb39) were generated by standard cloning procedures (see
4
Split Luciferase Assay for Cell Signaling
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Split luciferase reporter assays were carried out using the Promega NanoBiT system.
Briefly, cells were diluted in in HEPES buffered DMEM/F12 1:1 (Merck) supplemented with 10% fetal bovine serum and 2 mM L-glutamine to 50,000 cells/ml and plated at a density of 5000 cells per well (100 μl) in 96-well white clearbottom tissue culture treated plates (Corning 3610). Cells were grown for 24 h before transfection. Cells were transiently transfected with 50 ng each of the appropriate plasmids using FuGENE HD (Promega) at a FuGENE DNA ratio of 3:1.
After transfection cells were grown for a further 24 h before assay.
Plates were left to equilibrate at room temperature for 20 minutes before addition of NanoGlo live cell substrate (Promega) according to the manufacturer's instructions. After mixing at 800 rpm for 10 s, luminescence was assayed in a PHERAstar microplate reader (BMG LABTECH) at 25 °C using bottom optics and an averaging time of 10 s.
Briefly, cells were diluted in in HEPES buffered DMEM/F12 1:1 (Merck) supplemented with 10% fetal bovine serum and 2 mM L-glutamine to 50,000 cells/ml and plated at a density of 5000 cells per well (100 μl) in 96-well white clearbottom tissue culture treated plates (Corning 3610). Cells were grown for 24 h before transfection. Cells were transiently transfected with 50 ng each of the appropriate plasmids using FuGENE HD (Promega) at a FuGENE DNA ratio of 3:1.
After transfection cells were grown for a further 24 h before assay.
Plates were left to equilibrate at room temperature for 20 minutes before addition of NanoGlo live cell substrate (Promega) according to the manufacturer's instructions. After mixing at 800 rpm for 10 s, luminescence was assayed in a PHERAstar microplate reader (BMG LABTECH) at 25 °C using bottom optics and an averaging time of 10 s.
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