| Assay Method Information | |
| | Receptor Binding Assays |
| Description: | Receptor binding assays were performed by the Psychoactive Drug Screening Program (PDSP) at Chapel Hill, N.C. The assay protocol book can be accessed free of charge at: website pdsp.med.unc.edu/PDSP %20Protocols %20II %202013-03-28.pdf, which is incorporated by reference in it's entirety for all purposes. Briefly, Sig1Rs and Sig2Rs were sourced from homogenates of Guinea pig brains and rat livers, respectively. Assessment of Sig1R binding affinity was determined via competition binding assays with the radioligand [3H]-(+)-pentazocine. Sig2R binding affinity was determined through competition binding assays using the radioligand [3H]-ditolylguanidine in the presence of (+)-pentazocine to block Sig1R binding sites. For primary binding results, non-specific binding in the presence of 10 mM is set as 100% inhibition; total binding in the absence of haloperidol is set to 0% inhibition. The radioactivity in the presence of the test compound is calculated with the following equation and expressed as a percent inhibition: % inhibition=(sample CPM non-specific CPM)/Total CPM−non-specific CPM)×100. The normalization process is carried out in Prism or Excel. To determine secondary binding results, CPM/well are pooled and fitted to a three parameter logistic function for competition binding in Prism v 5.0 to determine IC50 values, which are converted to Ki according to the Cheng-Prusoff equation. |
| Affinity data for this assay | |
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