| Assay Method Information | |
| | ORL-1 GTPgammaS Binding Assay |
| Description: | Membranes from recombinant HEK-293 cells expressing the human opioid receptor-like (ORL-1) (Receptor Biology) were prepared by lysing cells in ice-cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Membranes were collected by centrifugation at 30,000xg for 15 min at 4° C., and pellets resuspended in hypotonic buffer to a final concentration of 1-3 mg/mL. Protein concentrations were determined using the BioRad protein assay reagent with bovine serum albumen as a standard. Aliquots of the ORL-1 receptor membranes were stored at -80° C. Functional binding assays were conducted as follows. ORL-1 membrane solution was prepared by sequentially adding final concentrations of 0.066 ug/uL ORL-1 membrane protein, 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]G IPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7. |
| Affinity data for this assay | |
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