Assay Method Information

Assay Name:  Radioligand Dose-Displacement Binding Assays (kappa)
Description:  Membranes from recombinant HEK-293 cells expressing the human κ opioid receptor (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 30,000×g for 15 min 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 κ 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 μg membrane protein (recombinant κ opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 μl binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4).
Affinity data for this assay
 

If you find an error in this entry please send us an E-mail