Proteomic Studies of Isolated Lipid Droplets from Bacteria, C. elegans, and Mammals - Methods in Cell Biology - page 6

1.2.5 The worms are homogenized by Polytron to release the cells into
the buffer suspension.
1.2.6 The cell suspension of worms is subjected to nitrogen bomb for
further homogenization.
1.2.7 The sample is then centrifuged at 1000

g for 10 min to remove

nucleus.
1.2.8 The steps 1.1.6-1.1.9 are performed.
Note : C. elegans cultured in liquid has less LDs.
1.3 Mammalian tissues or cells ( Fig. 1.1 C, CHO K2)
CHO K2 cells are cultured in DMEM plus 10% calf serum to 100%
confluence.
1.3.1 The cells are washed with cold PBS and scraped in Buffer A.
1.3.2 The skeletal muscle tissue is homogenized using a Polytron,
then followed by Dounce homogenizer. Liver tissue is
homogenized directly using the Dounce homogenizer.
1.3.3 The suspensions of step 1.3.2 or cultured mammalian cells of
step 1.3.1 are homogenized via nitrogen bomb.
1.3.4 The samples are then centrifuged at 1000

g for 10 min to

remove the nucleus.
1.3.5 Then steps 1.1.6-1.1.9 are performed.
Notes : Liver can be homogenized using Dounce. 250 mM sucrose in
Buffer A is utilized to protect intracellular organelles.
1.4 Collected LD fraction from top of gradient is washed with Buffer B
thrice or washed until no pellets are detectable.
Note : The pellets are contaminated membranes.
Step 2 Quality control of isolated LDs
Several criteria are needed to evaluate the quality of purified LDs.
2.1 Density less than 1 g/cm 3
The major composition of LDs is neutral lipids. LDs will float up to the
top of the gradient ( Fig. 1.2 A).
2.2 Ease of suspension (no apparent visible aggregation)
Since LDs are coated with proteins on the surface, the isolated LDs
should be relatively independent from each other. If some LDs are broken,
they tend to aggregate together. The aggregated LDs can be removed by
washing with Buffer B in such circumstances.
2.3 Milk-like consistency
Although different tissue or cells have different LDs, all isolated LD
suspensions share a common feature, a milk-like consistency ( Fig. 1.2 B).
2.4 Unique protein profile
LD proteins are precipitated using chloroform and acetone (1:1, vol/vol)
at room temperature. The LD proteins are then dissolved in SDS sample
buffer and separated by SDS-PAGE. The proteins in SDS-PAGE are either
stained with a silver stain kit or Colloid blue kit. The purity of isolated LDs
can be determined by unique protein profile of LDs compared with other