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[[Category:Metabolism]]
[[Category:Lipid Synthesis]]
[[Category:mTORC1]]
From PMID 20484008
Lipid synthesis and extraction. Cellular lipids were extracted essentially by the Folch method (6). Briefly, cells were incubated for 2 h at 37°C in the presence of [14C]acetic acid or [14C]glucose, washed once in cold PBS, and then lysed in 0.5 ml of ethanol. One milliliter of chloroform was added and mixed by vortexing. After addition of 0.5 ml of HCl (0.1 N), lysates were mixed by gentle inversion. Tubes were centrifuged at 500 g for 20 min. The upper phase and interface were discarded, and the organic phase was washed twice with 0.5 ml of water. Extracts were dried under N2, and the pellet was resuspended in the appropriate volume of chloroform-methanol (2:1). Extracts were separated by thin-layer chromatography with diethylether-hexane-glacial acetic acid (35:65:1, vol/vol/vol) as carrier solution on silica gel plates and exposed to film. Phospholipids and triacylglycerol were identified with standards.
[[Category:Lipid Synthesis]]
[[Category:mTORC1]]
From PMID 20484008
Lipid synthesis and extraction. Cellular lipids were extracted essentially by the Folch method (6). Briefly, cells were incubated for 2 h at 37°C in the presence of [14C]acetic acid or [14C]glucose, washed once in cold PBS, and then lysed in 0.5 ml of ethanol. One milliliter of chloroform was added and mixed by vortexing. After addition of 0.5 ml of HCl (0.1 N), lysates were mixed by gentle inversion. Tubes were centrifuged at 500 g for 20 min. The upper phase and interface were discarded, and the organic phase was washed twice with 0.5 ml of water. Extracts were dried under N2, and the pellet was resuspended in the appropriate volume of chloroform-methanol (2:1). Extracts were separated by thin-layer chromatography with diethylether-hexane-glacial acetic acid (35:65:1, vol/vol/vol) as carrier solution on silica gel plates and exposed to film. Phospholipids and triacylglycerol were identified with standards.
