| Assay Method Information | |
| | Binding Assay |
| Description: | The displacement of the labeled ligand by the test compound leads to a decreased signal. For the binding experiments 200 μL of membrane homogenate from one of the following protein amounts is used: hSSTR1 (40 μg/well); hSSTR2 (25 μg/well); hSSTR3 (1.5 μg/well); hSSTR4 (0.5 μg/well); hSSTR5 (25 μg/well). The homogenate is incubated with 0.05 nM of radioligand ([3-125I-Tyr]-Somatostatin-(1-14)) in addition to increasing concentrations of a test compound or vehicle (100% binding) in a total volume of 250 μL using a Hepes buffer (10 mM, EDTA 1 mM, MgCl2 5 mM, pH 7.6, BSA 0.5%, Bacitracin 0.003%, DMSO 1%) for 180 min at RT. The incubation is terminated by filtration with ice cold NaCl 0.9% through polyethyleneimine treated (0.3%) grade GF/B glass fiber filters using a cell harvester. The protein-bound radioactivity is measured in a suitable reader. The non-specific binding is defined as radioactivity bound in the presence of 1 μM Somatostatin-14 during the incubation period. The analysis of the concentration-binding curves is performed by computer-assisted nonlinear least square curve fitting method using the model of one receptor binding site. |
| Affinity data for this assay | |
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