Lipid Droplet Isolation Kit

Produktnummer ABIN5067556

Produktdetails

Applikation: Purification (Purif)

Proben: Cell Samples, Tissue Samples

Produktmerkmale: Lipid Droplet Isolation Kit isolates lipid droplets by simple gradient centrifugation, but circumvents the need for large sample sizes or ultracentrifugation. A lipid droplet source such as tissue or cultured cells is homogenized. A gradient is then created with the homogenate, and the material is centrifuged. The lipid droplets float to the top of the gradient and are recovered by carefully pipetting from the top of the gradient. Each kit provides sufficient reagents to isolate up to 50 preps based on a 50-100 mg tissue or cultured cell sample size.

Bestandteile:

1. Reagent A : One 10 mL bottle.
2. 10X Reagent B : One 7 mL bottle. 2

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Isolates lipid droplets from cells or tissues
• Uses a simple gradient centrifugation protocol
• No ultracentrifugation required

Protokoll: I. Isolation from Cultured Cells 7

1. Trypsinize 1.5-3 x 10 cells (roughly 50-100 mg) and resuspend in 10 mL of growth media in a 15 mL polypropylene tube.
2. Pellet cells at 1000 x g for 5 minutes.
3. Aspirate the media and wash cells with 10 mL of 1X PBS.
4. Pellet cells again at 1000 x g for 5 minutes.
5. Aspirate media and add 1 mL of 1X PBS.
6. Resuspend cells thoroughly and transfer to a 2 mL microcentrifuge tube.
7. Pellet cells again at 1000 x g for 5 minutes.
8. Aspirate 1X PBS and resuspend pellet thoroughly with 200 μL of Reagent A.
9. Incubate on ice for 10 minutes.
10. Add 800 μL of 1X Reagent B and mix well.
11. Incubate on ice for 10 minutes.
12. Homogenize the cells by passing them five times through a one inch 27 gauge needle attached to a 3 mL syringe.
13. Briefly spin the homogenate at 100 x g for 5 seconds 3
14. Carefully layer 600 μL of 1X Reagent B on top of the homogenate by dropwise addition taking care not to disturb the homogenate.
15. Spin the 2 mL microcentrifuge tube in a microcentrifuge for 3 hours at 18000-20000 x g at 4°C.
16. Carefully remove 270 μL (containing the floating lipid droplets) from the top of the tube and transfer to a fresh microcentrifuge tube.
17. Store lipid droplets at -80°C. II. Isolation from Tissue by Dounce Homogenization
    1. Weigh out 50-100 mg of tissue and mince into small pieces with a scalpel or scissors.
    2. Transfer minced tissue to a glass dounce.
    3. Add 200 μL of Reagent A.
    4. Incubate on ice for 10 minutes.
    5. Add 800 μL of 1X Reagent B.
    6. Incubate on ice for 10 minutes.
    7. Homogenize the tissue by performing 5 up and down strokes with the loose (A) pestle followed by 5 up and down strokes with the tight (B) pestle.
    8. Transfer 1 mL of the homogenate to a 2 mL microcentrifuge tube
    9. Carefully layer 600 μL of 1X Reagent B on top of the homogenate by dropwise addition taking care not to disturb the homogenate.
    10. Spin the 2 mL microcentrifuge tube in a microcentrifuge for 3 hours at 18000-20000 xg at 4°C.
    11. Carefully remove 270 μL (containing the floating lipid droplets) from the top of the tube and transfer to a fresh microcentrifuge tube.
    12. Store lipid droplets at -80°C.

Aufbereitung der Reagenzien:

1X Reagent B: Dilute 10X Reagent B to 1X with deionized water. Stir to homogeneity.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung: Store the entire kit at 4°C. To avoid possible leakage store bottles upright.

Antigendetails

Hintergrund: Lipid droplets are organelles that are rich in lipids, contain a lipid rich core, and are surrounded by a phospholipid monolayer as well as outer lipid droplet associated proteins. Lipid droplets are commonly found in adipose tissue of animals, although they are found in all eukaryotes. Lipid droplets function to regulate the hydrolysis and storage of neutral lipids and also serve as storage for cholesterol and acyl- glycerols used to form and maintain cellular membranes. Beyond the function of lipid and cholesterol storage, lipid droplet organelles have been more recently associated with inflammatory responses, obesity, atherosclerosis, and cancer. Lipid droplets have been shown to protect against lipotoxicity in non-adipocytes by storing fatty acids as neutral triacylglycerol. Lipid droplets also aid in protein binding and degradation and have been demonstrated to be used by pathogens such as dengue virus and hepatitis C Virus.