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New lipid extraction method for the determination of phospholipase D activity with improved recovery and sensitivity
New lipid extraction method for the determination of phospholipase D activity with improved recovery and sensitivity
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机译：测定脂质磷脂酶D活性的新脂质提取方法，提高了回收率和灵敏度
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摘要
PURPOSE: Provided is a lipid-extracting method for verifying the activity of phospholipase D, which extracts only phosphatidyl ethanol(PEt) or phosphatidyl butanol(PBt) selectively under the acidic condition, thereby improving the degree of recovery and sensitivity. CONSTITUTION: The lipid extracting method comprises the steps of: (1) obtaining cell lines by centrifugation, emulsifying with 500-1000 microliter of water, and adding 50-100 microliter of 2N hydrochloric acid solution and mixing; (2) adding 600-1200 microliter of ethyl acetate to the mixture solution of the steps (1) and stirring for 5-15min at 1500-2500rpm and leaving alone at ordinary temperature; (3) centrifuging the mixture of the step (2) at ordinary temperature and recovering an upper organic solvent layer; (4) adding 500-1000 microliter of ethyl acetate to the rest water solution layer of the step (3) and stirring for 5-15min at 1500-2500rpm and then leaving alone; (5) centrifuging the mixture of the step (4) at ordinary temperature and taking an upper organic solvent layer and mixing with the organic solvent layer of the step (3); (6) leaving the mixture of the step (5) alone at -70 to -20deg.C for 2-14 hours and then performing centrifugation at ordinary temperature and removing a lower water solution layer; (7) drying the organic solvent layer of the step (6) and dissolving in 50-300 microliter of a mixture solution of chloroform and methanol and analyzing by a thin layer chromatography(TLC).
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