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<br> The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse GLP-1. During detergent addition, a custom rabbit anti-Gαs/olf polyclonal antibody created like sc-383 (Santa Cruz Biotechnology Inc.), used to mimic its properties, and 0.25 mg of anti-rabbit IgG polyvinyltoluene scintillant beads (RPNQ0016, PerkinElmer) were also added to the mixture. Recruitment of ARRB2 to activated receptors occurred at 37°C for 90 minutes, followed by cell lysis by the addition of 10 μL detection mixture containing β-galactosidase 93-0001 substrate to quantitate functional enzyme fragment complementation. Agonist-stimulated protein-to-protein interaction of 2 fusion molecules were detected with Nano-Glo substrate and standard luminescent detection. BRET was performed with the Nano-Glo substrate (N1662) as donor and NanoBRET 618 ligand as acceptor. Internalization was performed as described above, [https://wiki-staging.jgtitleco.com/index.php?title=Introducing_ColonBroom:_The_Revolutionary_GLP-1_Supplement_For_Optimal_Colon_Health appetite and satiety support] cells were then fixed with 4% paraformaldehyde and washed with PBS. Then, the cells were stimulated with 37°C preheated ligand, and internalization was measured every 3 minutes for 60 minutes at 37°C by an EnVision plate reader. The following day, the media was removed and the tagged receptors were labeled with 100 nM Tag-Lite SNAP-Lumi4-Tb (donor, Cisbio), in OptiMEM for 75 minutes at 37°C. Afterward, the cells were washed with internalization buffer (HBBS supplemented with 1 mM CaCl2, 2.5 mM MgCl2, 20 mM HEPES,  [https://wiki.mubk.top/index.php?title=User:WilliamByron6 appetite and satiety support] and 0.1% Pluronic F-68, pH 7.4) followed by addition of 100 μM preheated fluorescein-O’-acetic acid (acceptor, Sigma-Aldrich).<br><br><br><br> Culture medium was comprised of Dulbecco’s Modified Eagle Medium (DMEM) containing 25 mM glucose, 10% fetal bovine serum (FBS), 100 units ml−1 penicillin G, and 100 μg/ml streptomycin. 100 nL ligand via acoustic direct dilution, as mentioned above, prior to prewarming at 37°C. Cell vials were thawed and added at equal volume in respective assay medium as previously stated. The plate was placed in a 37°C incubator for 5 minutes prior to ligand addition to adjust the temperature. Chemiluminescent signal was developed for 60 minutes at room temperature and quantified using an EnVision plate reader. After 48 hours, cells were removed from flasks using enzyme-free dissociation buffer (13151014, Thermo Fisher Scientific), followed by trituration, brief centrifugation (500g, 5 minutes, room temperature) and filtration (40 μm cell strainer, Thermo Fisher Scientific). Following 48-hour shaking, cells were pelleted and resuspended in assay buffer (1× PBS, 0.1% w/v bovine casein). Ligand (10×) was titrated by manual serial dilution in DMSO followed by step-down into assay buffer. Energy minimizations were performed to relieve unfavourable contacts in the system, followed by equilibration steps of 50 ns in total to equilibrate the lipid bilayer and the solvent with restraints on the main chain or Cα atoms of GLP-1R.<br><br><br><br> Where R is receptor (e.g., GIPR or GLP-1R), A is albumin, and L is a ligand that can bind to both the receptor and albumin (e.g., acylated GIPR/GLP-1R agonist). Kinetic cAMP assays. Kinetic cAMP assays were performed in low-density GIPR and GLP-1R cell clones transfected with the Glosensor 22F vector (Promega) (53).