BRET assays were performed similarly to previous work (94, 95). HEK 293T cells (CRL-3216, American Type Culture Collection) were seeded on six-well plates coated with gelatin at a density of ~400,000 cells per well for transfection on the next day. Cells were transfected with the BRET donor and acceptor components GRK3ct-nLuc (0.2 μg), Venus(ct)-Gβ1 (0.2 μg), and Venus(nt)-Gγ2 (0.2 μg), as well as with plasmids encoding the appropriate G proteins and GPCRs. For experiments with Gi, cells were cotransfected with 1 μg of plasmid encoding Gαi3 and 0.2 μg of plasmid encoding α2-AR. For Gq experiments, plasmids encoding Gαq (1 μg) and M3R (0.2 μg) were used. Cells were also cotransfected with either 0.5 or 1 μg of plasmid encoding tagged, full-length RGS protein (or empty vector for controls) with the BRET components and plasmids encoding G protein and GPCR. For RGS7, cells were also cotransfected with 1 μg each of plasmids encoding R7BP and Gβ5 to reconstitute full GAP activity (77). Empty pcDNA3.1 vector was used to equalize the total amount of DNA for each transfection. Transfections were performed with the calcium phosphate method. About 16 to 24 hours after transfection, cells were washed once in room temperature phosphate-buffered saline (PBS) and gently harvested from the plate surface in 1 ml of PBS by scraping. Recovered cells were centrifuged for 5 min at 550g and resuspended at a concentration of ~1 × 106 cells/ml in BRET buffer [140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.37 mM NaH2PO4, 24 mM NaHCO3, 10 mM Hepes, and 0.1% glucose (pH 7.4)]. For BRET measurements, about 15,000 cells were added to a white opaque 96-well plate (OptiPlate, PerkinElmer) and mixed with Nano-Glo nanoluciferase substrate (Promega, final dilution of 1:200) in a final volume of 100 μl for 2 min before measuring luminescence. Baseline luminescence was recorded for 30 s before the addition of 1 μM brimonidine agonist (for the Gi studies) or 100 μM carbachol (for the Gq studies). After 60 s of monitoring the BRET response to stimulation, 50 μM yohimbine (antagonist used for the Gi studies) or 100 μM atropine (antagonist used for the Gq studies) was added to measure the off-rate kinetics. Luminescence signals were simultaneously measured at 460 ± 40 nm and 535 ± 35 nm using a POLARstar plate reader (BMG Labtech) at 28°C every 0.24 s. BRET values represent the ratio between the 535 ± 35–nm and 460 ± 40–nm emission intensities (raw BRET ratio). For the calculation of response amplitudes, the difference between the raw BRET ratio before and 60 s after agonist stimulation was calculated. For presentation and the calculation of off-rate results, the maximal deactivation value reached after the addition of antagonist (plateau signal) was subtracted from the raw BRET ratio of each time point (recovery corrected ΔBRET), and each resulting value was scaled to the percentage of the maximal agonist-induced activation value directly before the addition of antagonist (final result “% Max BRET”). Off-rates were determined by plotting the recovery-corrected ΔBRET values for 150 s after the addition of antagonist (starting at 90 s, Y0) and fitting to a one-phase decay model to determine the rate (k), where the plateau is the maximal deactivation valueY=(Y0plateau)×e(k×X)+plateau

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.