Biology Reference
In-Depth Information
Table 7.1
Lipid Droplet Isolation from Mouse Livers
1. Harvest livers quickly and carefully
2. Clean livers from any other tissues
3. Rinse livers thoroughly in ice-cold PBS
4. Homogenize livers by soft homogenization in HM on ice
5. Transfer homogenates to 15 ml tubes
6. Centrifuge at 500
g for 10 min at 4
C
7. Spin the supernatant at 15,000
g for 10 min at 4
C
8. Recover fat cakes into new clean tubes
9. Wash twice at 15,000
g for 10 min at 4
C
10. Dilute fat cakes with 1/3 volume of ice-cold HM containing 60% sucrose (final
concentration 20% sucrose)
11. Disperse aggregates of LDs by gently pipetting
12. Layer the diluted aggregates carefully at the bottom of ultracentrifuge tubes
13. Overlay these tubes with double the volume HM containing 5% sucrose
14. Carefully overlay the tubes with same volume of HM
15. Ultracentrifuge tubes at 28,000
g for 30 min
16. Recover LDs carefully (use a pipet or a slicer) into Eppendorf tubes
17. Analysis
(modified from
Brasaemle & Wolins, 2006
)
leftover adipose tissue. Individual livers are transferred to a beaker containing ice-
cold PBS and are thoroughly rinsed. Then, livers are weighed and immediately sub-
jected to soft homogenization on ice in HM (20% homogenate, w/v) by 10 gentle
strokes with a motor-driven Potter-Elvehjem homogenizer set at a speed of 3. This
step is crucial to preserve LD integrity and to prevent damage to intracellular organ-
elles (such as the ER, which would otherwise leak lumenal contents including VLDL
and LLDs that would contaminate CLD preparations). Homogenates are transferred
to 15 ml tubes and centrifuged 10 min at 500
g
,4
C. The supernatant containing
the floating fat layer (fat cake) is then spun at 15,000
g
for 10 min to remove mito-
chondria and to further allow fat cake separation. Fat cakes are then recovered into new
tubes and washed twice with ice-cold HM at 15,000
g
for 10 min. Recovered fat
cakes are then diluted with 1:3 (v/v) of ice-cold HM containing 60% sucrose to yield
a final 20% sucrose-adjusted homogenate. Aggregates of LDs should be finely and
thoroughly dispersed by gentle pipetting. It is highly recommended to use a pipet
tip with a wide opening. Diluted CLD aggregates are now carefully layered at the bot-
tom of ultracentrifuge tubes and overlayed with double the volume HM containing 5%
sucrose. These tubes are now carefully overlayed with same volume of HM. Tubes
should be ultracentrifuged at 28,000
g
for 30 min. After ultracentrifugation, fat cakes
are carefully recovered (using a pipet or a slicer) and kept in Eppendorf tubes for fur-
ther analysis. This step could be repeated after further dilution of recovered fat cake
with 60% sucrose in order to get a highly enriched (purified) CLD fraction.
After isolation, recovery and purity of LDs should be evaluated by SDS-PAGE
followed by immunoblotting (see below how to solubilize CLD-associated proteins).
