Abstract
The principle of selective elution from a solid phase has been exploited to develop an assay for the determination of squalene biosynthesis in rat liver homogenates. Using either [1- 14C]isopentenyl diphosphate as a precursor for squalene or [2- 14C]farnesyl diphosphate as a direct substrate of squalene synthase, the production of radiolabeled squalene is determined after adsorption of assay mixtures onto silica gel thin-layer chromatography sheets and selective elution of the diphosphate precursors into a solution of sodium dodecyl sulfate at alkaline pH. The use of [2- 14C]farnesyl diphosphate, and of an endogenous oxygen consumption system (ascorbate/ascorbate oxidase) to prevent further metabolism of squalene, allows the method to be applied as a dedicated assay for squalene synthase activity. The assay has been developed in microtiter plate format and may be deployed either in a quantitative, low-throughput mode or in a qualitative, high-throughput mode. The latter is suitable for screening to aid in the discovery of new inhibitors of squalene synthase.
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