| Assay Method Information | |
| | Human Sortilin Binding Assay |
| Description: | The compound affinity was determined by measuring the inhibition of 3H-neurotensin and/or 125I-neurotensin binding to human full length sortilin using a Scintillation Proximity Assay (SPA) format.The human sortilin SPA binding assay was performed in a total volume of 40 μl in 50 mM HEPES pH 7.4 assay buffer containing 100 mM NaCl, 2.0 mM CaCl2, 0.1% (v:v) BSA and 0.1% (v:v)Tween-20. Varying concentration of test compounds where pre-incubated for 30 min at RT with 150 nM of c-terminal hexahistidine- human full length Sortilin (RD Systems). 5 nM [3H]-Neurotensin or 0.35 nM [125I]-Neurotensin was added as radioligand and nonspecific binding defined as the binding observed in the presence 20 μM of human Neurotensin. 0.1 mg PVT copper HIS-tag imaging beads (Perkin Elmer) was added and the plate was agitated at 400 rpm in the dark for 1 hr. The SPA beads were allowed a minimum 2 hours settling time before the plate was read on a Hidex Sense 425-301 scintillation reader with a 45 sec exposure time. Concentration-response evaluation of test compounds was performed with 8 different concentrations of compound (covering 3 decades). Using assay high and low controls (0 μM and 20 μM human neurotensin, respectively), relative IC50 values were calculated by nonlinear regression analysis using the sigmoid concentration-response (variable slope) fitting option available in GraphPad Prism 9.0. The compound potency results were converted to inhibition constant Ki values (nM) derived from calculated IC50 values converted to Ki values using the Cheng-Prusoff equation (Ki=IC50/(I+(L/Kd))). Kd for human neurotensin was experimentally determined by saturation binding as 374 nM (n=2). |
| Affinity data for this assay | |
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