| Assay Method Information | |
| | Inhibition of Pro Part of ProNGF to hSortilin |
| Description: | The pro Sortilin assay was performed in total volume of 20 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 incubated for 15 min at RT with 50 nM of 6his-Sortilin and 6 nM of pro-GST. 4 nM of anti-GST-Eu and 25 nM of anti-His-XL665 were added together with KF (final concentration 200 mM) and, following 150 min incubation at room temperature in the dark. the fluorescence was measured with excitation of 320 nm and dual emission of 665 and 615 nm on the an envision reader (Perkin Elmer). Signal was expressed in terms of HTRF ratio (fluorescence intensity at 665 nm/fluorescence intensity at 615 nm×10,000). Inhibition of binding of proGST to Sortilin was expressed as a percent inhibition of the control response to 20 μM of Neurotensin (100% inhibition) relative to a buffer basal control (0% inhibition). IC50 values were calculated by nonlinear regression using the sigmoid concentration-response (variable slope) using Xlfit 4 (IDBS, UK). |
| Affinity data for this assay | |
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