| Assay Method Information | |
| | mSortilin Affinity Assay |
| Description: | The Sortilin assay was performed in total volume of 40 μl in 50 mM HEPES pH 7.4 assay buffer containing 100 mM NaCl, 2.0 mM CaCl2, 0.1% BSA and 0.1% Tween-20. Varying concentration of compounds where pre-incubated for 30 min 10 at RT with 50 nM of 6his-Sortilin. 5 nM [3H]-Neurotensin was added as radioligand and nonspecific binding defined as the binding in the presence 20 μM of Neurotensin. Ni chelate imaging beads (Perkin elmer) were added and the plate was slowly shaken in the dark for 60 min. The imaging beads were allowed a minimum 6 hours settling time before the plate was read on a ViewLux with 360 sec exposure time. Dose-response evaluation of compounds was performed with 10 concentrations of drugs (covering 3 decades). IC50 values were calculated by nonlinear regression using the sigmoid concentration-response (variable slope) using Xlfit 4 (IDBS, UK). The results were given as Ki values (nM) derived from computer-fitted IC50 values converted to Ki values using the Cheng-Prusoff equation (Ki=20 IC50/(1+(L/Kd))). Kd for Neurotensin was determined to 100 nM. |
| Affinity data for this assay | |
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