Assay Method Information |
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| Binding Assay |
Description: | Receptor binding assay: Membranes were prepared from CHO cells expressing S1P1 or S1P3 for use in ligand and 35S-GTPγS binding studies. Cells were suspended in 50 mM TRIS, pH 7.4, 2 mM EDTA, 250 mM Sucrose (buffer A) and 1 Complete protease inhibitor cocktail (Roche), and disrupted at 4 C. by N2 decompression using a cell disruption bomb (Parr Instrument). Following centrifugation at 1000 RPM for 10 min at 4 C., the supernatant was suspended in buffer A and centrifuged again at 19000 RPM for 60 min at 4 C. The pellet was then suspended in 10 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM Sucrose (Buffer B), and 1xComplete EDTA-free protease inhibitor cocktail and homogenized using a potter. Membranes were flash frozen in liquid N2 and stored at −80 C. [33P]sphingosine 1-phosphate (3000 Ci/mmol; American Radiolabeled Chemicals, Inc.) was added to test compounds in DMSO. |
Affinity data for this assay | |
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