| Assay Method Information | |
| | In vitro Exogenous Substrate Assay |
| Description: | Briefly, PLD activity was measured as the release of free [3H]-choline from [choline-methyl-3H] dipalmitoylphosphatidylcholine ([3H]-DPPC). 3−50 nM PLD1 or PLD2 was reconstituted with phospholipid vesicle substrates composed of 10 μMdipalmitoyl-PC, 100 μM PE (bovine liver), 6.2 μM PIP2 (porcine brain), 1.4 μM cholesterol, and 2.5 μCi [3H]-DPPC. Lipid solutions were dried under a gentle stream of nitrogen and resuspended in 100 mM HEPES (pH 7.5), 160 mM KCl, 6 mM EGTA, and 0.2 mM DTT. Small unilamellar vesicles were prepared by bath sonication (2 × 2 min intervals at 80 W). All assays were performed at 37 °C on agitation for30 min in 50 mM HEPES, pH 7.5, 80 mM KCl, 3 mM EGTA, 0.1 mM DTT, 3.6 mM MgCl2, 3.6 mM CaCl2, and 10 μM GTPγS. Reactions were stopped with the addition of trichloroacetic acid and bovine serum albumin. Free [3H]-choline was separated from precipitated lipids and proteins by centrifugation and was analyzed by liquidscintillatio |
| Affinity data for this assay | |
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