| Assay Method Information | |
| | Binding Assay |
| Description: | Kippa-Opioid Receptor Binding Assay Procedures: Membranes from recombinant HEK-293 cells expressing the human kippa opioid receptor (kippa) (cloned in house) 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 mM at 4 C. and pellets were 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 standard. Aliquots of kippa receptor membranes were stored at -80 C.Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 ug membrane protein (recombinant lc opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 ul binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). |
| Affinity data for this assay | |
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