DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin; Sigma Aldrich, St Louis, USA), morphine, M3G and CTOP ([H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2]; Sigma Aldrich, St Louis, USA) were used in [35S]-GTPγS binding assay to measure G protein activation following receptor stimulation. Brain membranes were prepared from WT and conventional KO mice as previously described21 (link). Membrane preparations from brain were incubated for 1 h at 25 °C with increasing concentrations (10−9 to 10−4 M) of agonists (DAMGO, Morphine or M3G) in the assay buffer containing 30 µM GDP and 0.1 nM [35S]-GTPγS (NEG030H, PerkinElmer, Courtaboeuf, France). Basal [35S]-GTPγS binding was determined in the absence of agonist, and non-specific binding by replacing [35S]-GTPγS by cold GTPγS. For the experiment with the mu antagonist CTOP, WT brain membranes were incubated with a fixed dose of DAMGO, morphine and M3G (10−4M) and with increasing concentrations of CTOP (0 to 30 μM) to assess the specific activation of mu-receptor by these agonists. Stimulated specific binding was converted in percentage of basal specific binding, defined as 100%. Data were analyzed using Prism 6 Graphpad software. Four to ten independent assays were performed on three distinct membrane preparations per genotype. Stimulation (%), EC50s and IC50s were calculated for each experiment and averaged.
Damgo
Manufactured by PerkinElmer
Sourced in France
DAMGO is a synthetic opioid peptide primarily used in scientific research. It functions as a highly selective mu-opioid receptor agonist. The core purpose of DAMGO is to serve as a tool for studying opioid receptor pharmacology and signaling in various experimental models.
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Lab products found in correlation
2 protocols using damgo
1
Measuring G Protein Activation in Brain Membranes
2
Saturation Binding Assay for HA-MOR and FLAG-CCKBR
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HEK293 cells stably expressing HA-MOR or co-expressing HA-MOR and FLAG-CCKBR were harvested and lysed using the binding buffer containing 5 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 5 mM EGTA at 4 °C for 30 min. The harvested cells were centrifuged at 40,000×g for 20 min at 4 °C, and the pellet was homogenized by filtering through a needle. The homogenate was then centrifuged at 40,000×g for 20 min at 4 °C. The pellet was resuspended in the binding buffer and used as the membrane preparation for the radio-ligand-binding assay. Protein concentration in the prepared membrane was measured by the Coomassie Brilliant Blue method. For the saturation-binding analysis, cell membranes (50–80 μg of protein per assay tube) were incubated with gradient concentrations of [3H]-[D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; PerkinElmer) for 1 h at 37 °C. Nonspecific binding was determined by adding 100 nM DAMGO (Sigma-Aldrich) to the reaction mixture. The binding reaction was terminated by the rapid filtration through glass microfiber filters. Filters were washed with ice-cold binding buffer three times, and the bound radioactivity was measured using a liquid scintillation counter. The Prism program (GraphPad Software, La Jolla, CA) was used to analyze the data derived from the saturation-binding assays and obtain Bmax and KD values.
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