期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 146, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/59290
关键词
Biology; Issue 146; Lipid droplet isolation; liver; perilipin 2; endoplasmic reticulum; sucrose gradient; isotonic extraction buffer
资金
- NIH/NIAAA [R01 AA026302-01, K08-AA021424, F32-AA024347]
- Robert Wood Johnson Foundation
- Harold Amos Medical Faculty Development Award [7158]
- IDOM DRC Pilot Award [P30 DK019525]
- NIH [P30-DK050306]
Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver. Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.
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