HEK293 cells were cultured in 6-well plates and transiently co-transfected with β-arrestin 2-Nluc and [WT]D2R-HT, [Gprot]D2R-HT, [βarr]D2R-HT or [A3]D2R-HT. After overnight incubation, cells were plated into white 96-well plates (Costar) and cultured in Opti-MEM (Life Technologies) containing 4% FBS. HaloTag containing samples were labeled with HaloTag 618 ligand (Promega) at 100 nM of final concentration and incubated at 37°C overnight. The Nano-Glo substrate was added into each well following manufacturer’s instruction (Promega). Donor emission (460 nm) and acceptor emission (610 nm) were measured as a basal signal by SpectraMax M5 spectrometer (Molecular Devices). The chemicals were then added as required and the plate was read.
Halotag 618 ligand
Manufactured by Promega
The HaloTag 618 ligand is a fluorescent dye that can be used to label and detect HaloTag fusion proteins. The HaloTag 618 ligand binds covalently to the HaloTag protein, allowing the target protein to be visualized in live or fixed cells.
Automatically generated - may contain errors
Lab products found in correlation
White 96 well plate, by Corning (1 mentions)
Spectramax m5 spectrometer, by Molecular Devices (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Furimazine, by Promega (1 mentions)
Synergy neo2 plate reader, by Agilent Technologies (1 mentions)
Nanobret nano glo substrate, by Promega (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Fugene hd, by Promega (1 mentions)
4 protocols using halotag 618 ligand
1
BRET-based D2 Receptor Characterization
2
CXCR4-Arrestin Recruitment and Internalization Assay
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HEK293AD cells were seeded in 10-cm cell culture dishes at a density of 2 × 106. For arrestin recruitment assay, cells were cotransfected with 1000 ng of CXCR4-nLuc and 1,000 ng of human β-arrestin2-HaloTag. For internalization assay, a plasmid construct bearing two HaloTags, separated by a repetitive rigid linker, and an N-terminal Lyn-derived sequence (GCIKSKRKDK) and a C-terminal farnesylation sequence (KKKSKTKCVIM) for plasma membrane localization was used. Then 1,500 ng of this plasmid was cotransfected with 500 ng of CXCR4-nLuc 24 h after seeding, and the cells were transferred into a while-walled, white-bottom 96-well plate, at a density of 105 cells per well, and cells were labeled for at least 16 h with HaloTag 618 Ligand (Promega); 16 h to 24 h after reseeding, medium was removed, and cells were washed with PBS and then supplemented with 90 μL HBSS containing furimazine (Promega) (1:1,000 vol/vol). After 10 min incubation at 37 °C, measurement was performed at 37 °C using a Synergy Neo2 Plate Reader (Biotek) using the NanoBRET filter set.
3
CK1α Inhibitor Screening in HEK293 Cells
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CSNK1A1CK1α-HiBiT HEK293 LgBiT stable cells weretransfected with a HaloTag-CRBN vector (Promega, Cat. #N269A) using Fugene HD (Promega, Cat. #E2311). Following overnight incubation, cells were replated in OptiMEM I Reduced Serum Medium, no phenol red (Gibco, Cat. #11058021) supplemented with 4% FBS, and HaloTag 618 ligand (Promega, Cat. #G9801). Plates were incubated overnight at 37 °C with 5% CO2. On day 3, a dose–response curve was generated by performing 3-fold dilutions of each compound including a DMSO-only control in OptiMEM I Reduced Serum Medium, no phenol red supplemented with 4% FBS. Cells were treated with MG132 (Selleckchem, Cat. #S2619) for 30 min before being treated with the dilution series of compounds. Plates were incubated for 2 h at 37 °C with 5% CO2. After 2 h, plates were read using NanoBRET Nano-Glo substrate (Promega #N1573) on a GloMax Discover following the manufacturer’s instructions. No ligand controls were subtracted from each sample and normalized to the no compound DMSO control to obtain Fractional BRET values. EC50 values were calculated for each compound curve.
4
Receptor Activation Quantification in Cells
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Transfected cells had media replaced with DMEM with 10% FBS containing 100 nM HaloTag 618 ligand (Promega) and were incubated for 5 h at 37 °C with 5% CO2. Media was then replaced with HBSS with or without 5 nM IL-23 and the cells were incubated for a further hour in the absence of CO2. Following this 5 μL of HBSS with 77 μM furimizine was added to each well and the BRET signal was measured on a PheraStar FS plate reader using 460 ± 80 nm and >610 long pass filters.
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