James1689 - 29-12-2025 at 09:46 AM
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-G_s/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 _L detection mixture containing _-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, and 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, and 0.1%
Pluronic F-68, pH 7.4) followed by addition of 100 _M preheated fluorescein-O_-acetic acid (acceptor, Sigma-Aldrich).
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