| Assay Method Information | |
| | Homologous Competition Assay |
| Description: | Radioligand binding assays were performed as previously detailed [Yan et al., Biochemistry, 48:6898-6908]. In brief, crude cell membranes were prepared by lysing transfected HEK-T cells in binding buffer (50 mM Tris-Cl, 10 mM MgCl2, and 0.1 mM EDTA at pH 7.40) and centrifugation at 30,000 × g for 20 min. Membrane pellets were then flash-frozen in liquid nitrogen and stored at -80 °C until use in binding assays. Binding assays were conducted in total volumes of 0.25 ml in 96-well plates in a binding buffer containing 0.3-0.6 nM [3H]diprenorphine for a total of 60 min at room temperature. Assays were terminated by harvesting over 0.3% polyethyleneimine-treated, 96-well filter mats using a 96-well Filtermate harvester and three quick washes with ice-cold binding buffer. Diprenorphine Kd and receptor expression levels (Bmax) for the different mutants were determined using homologous competition binding. |
| Affinity data for this assay | |
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