| Assay Method Information | |
| | Human Kisspeptin Binding Assay Protocol |
| Description: | The human kisspeptin membrane binding assay utilizes the following components: radiolabel [125I]-metastin (45-54) (human) (Perkinhmer), crude Flp-In T-Rex 293 kisspeptin membranes, and competing small molecule and peptide ligands. The assay is initiated by combining in assay buffer (50 mM TrisHCl, 5 mM MgCl2, 2 mM EGTA, 10 mM CaCl2, 0.1% BSA, pH 7.4) a dose response of competing ligand (final concentrations are typically 0-10,000 nM), 5-10 μg of Flp-In T-Rex 293 kisspeptin membranes, and 0.2 n M [125I]-metastin (45-54) in a 96-well assay plate and allowed to incubate 90 minutes at room temperature. Assay contents are then filtered through unifilter CF/C: microplates (PerkinElmer) pre-soaked with 0.5% BSA and washed with 9×400 μL of cold wash buffer (10 mM HEPES, 500 mM NaCl, 0.1% Tween-20, pH7.4). Assay plates are read using a Microbeta2 (PerkinElmer) and Ki values for compounds are determined using a GraphPad Prism 9.3 non-linear regression analysis. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |