Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA169110

Local Sample ID:	10
Subject ID:	SU001897
Subject Type:	Cultured cells
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Not applicable

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Sample Preparation:

Sampleprep ID:	SP001903
Sampleprep Summary:	Lipid and small-molecule metabolite extraction was performed using a methyl tert-butyl ether (MTBE)/methanol extraction protocol. Briefly, harvested neurons were homogenized in ice-cold methanol. Following homogenization, samples were incubated in 1200 ul of MTBE for 1 hr at room temperature to separate organic-soluble lipids from aqueous-soluble lipids and other small-molecules. Finally, 360 ul of ultrapure water was added (for a final ratio of 3:1:0.9 MTBE:methanol:water) to resolve the two liquid phases, and each samples were centrifuged at 10,000 x g for 10 min. For this experiment, the upper organic phase was collected from each sample and stored in a separate tube, and the remaining protein pellets were resuspended in 25 mM ammonium bicarbonate, pH 8, with 2.5% SDS. A BCA protein assay was performed on each protein fraction, and the organic phase was normalized to their protein concentration equivalent with 100% methanol. All samples were then stored at -80°C prior to analysis.

Sampleprep ID:

SP001903

Sampleprep Summary:

Lipid and small-molecule metabolite extraction was performed using a methyl tert-butyl ether (MTBE)/methanol extraction protocol. Briefly, harvested neurons were homogenized in ice-cold methanol. Following homogenization, samples were incubated in 1200 ul of MTBE for 1 hr at room temperature to separate organic-soluble lipids from aqueous-soluble lipids and other small-molecules. Finally, 360 ul of ultrapure water was added (for a final ratio of 3:1:0.9 MTBE:methanol:water) to resolve the two liquid phases, and each samples were centrifuged at 10,000 x g for 10 min. For this experiment, the upper organic phase was collected from each sample and stored in a separate tube, and the remaining protein pellets were resuspended in 25 mM ammonium bicarbonate, pH 8, with 2.5% SDS. A BCA protein assay was performed on each protein fraction, and the organic phase was normalized to their protein concentration equivalent with 100% methanol. All samples were then stored at -80°C prior to analysis.