| Assay Method Information | |
| | Tag-Lite Tachykinin NK-1 receptor binding assay |
| Description: | Experimental procedure for competitive binding (Ki determination): Perform CisBio's Tag-Lite Tachykinin NK-1 binding assay according to manufacturer's protocol. Briefly, prepare each test compound in 8 points of concentration. Dilute compound with 1×TLB to an initial concentration of 40 nM (C1). Starting with the C1 solution (40 nM), prepare ⅕ serial dilutions in 1×TLB by adding 10 μL C1 to 40 μL of 1×TLB, mix gently and repeat the ⅕ serial dilutions to prepare C2, C3, C4, C5, C6, C7, C8 solutions. For competition dose-response of compounds, the optimal fluorescent ligand concentration is the one that allows 50% Ka of receptor binding. Add 5 μL of fluorescent ligand stock solution (10000 nM) to 1245 μL of 1×TLB, mix gently to obtain the 40 nM working solution. Competitive binding assay was run in duplicate for all assay points. Combine 10 μL labeled cells into each well; 5 μL compound dilutions (C1-C8) into each appropriate well; repeat for each compound tested, and 5 μL fluorescent ligand into each well. Incubate 2 h at room temperature, read the Signal665nm and Signal620nm using Evision. Data analysis was performed by calculating the Ratio following the formula below, the competitive binding data for each compound was analyzed with GraphPad Prism 5.0, and IC50 value was calculated for each compound. |
| Affinity data for this assay | |
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