| Assay Method Information | |
| | GTPgammaS Functional Assay (kappa) |
| Description: | Membranes from recombinant HEK-293 cells, CHO cells expressing the recombinant human kappa opioid receptor (kappa) 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. Functional [35S]GTPgammaS binding assays were conducted as follows. kappa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kappa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2,20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B. |
| Affinity data for this assay | |
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