| Assay Method Information | |
| | Scintillation Proximity Assay |
| Description: | Compounds are assessed for the ability to bind to FLAP in a binding assay that measures compound-specific displacement of an iodinated (125I) FLAP inhibitor via a Scintillation Proximity Assay format (adapted from S. Charleson et al., Mol. Pharmacol., 1992, 41, 873-879). Cell pellets produced from sf9 insect cells expressing recombinant human FLAP protein are resuspended in buffer A [15 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.3 mM EDTA, 1 mM PMSF]. The cells are lysed with a Dounce homogenizer and the material is centrifuged at 10,000xg for 10 minutes. The supernatant is then collected and centrifuged at 100,000xg for 60 minutes. To prepare membrane protein for an assay, an aliquot of the 100,000xg pellet is resuspended in 1 ml of buffer A, Dounce homogenized, and finally subjected to polytron mixing (30 seconds). Membrane protein (25 ul, 5 ug) is mixed with WGA SPA beads (Amersham) and stirred for 1 h. To an assay plate (Perkin Elmer FlexiPlate) is added 25 ul of test compound. |
| Affinity data for this assay | |
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