Biology Reference
In-Depth Information
FIGURE 1.1
Protein profile of isolated lipid droplets (LDs) from three species. Proteins extracted from
isolated LDs (LD), total membrane (TM), cytosol (Cyto), and post nuclear supernatant (PNS)
fractions of RHA 1 (A), Caenorhabditis elegans (B), CHO K2 (C) are loaded by equal amount
and separated in SDS-PAGE and stained by Colloidal blue (A and B) or silver staining kit (C).
LDs and PNS are obtained using methods described in
Section 3
. TM and Cyto are prepared
and described in previous studies. Briefly, 1 ml PNS is placed in a 9.5
38 mm
microfuge tube and centrifuged at 345,287
g for 10 min in a Beckman Optima Max
ultracentrifuge using a TLA 100.3 rotor at 4
C. The middle part of clean solution is collected
as Cyto fraction and the pellet is treated as TM fraction. Cyto fraction is directly mixed with 2
SDS sample buffer. TM pellet is washed twice with 1 ml Buffer B, and proteins are
precipitated by 7.2% trichloroacetic acid, washed with 1 ml 100% acetone three times,
and dissolved in SDS sample buffer.
(A) is reproduced from
Ding et al. (2012)
. (B) is reproduced from
Zhang et al. (2012)
. (C) is reproduced
from
Bartz, Zehmer, et al. (2007)
.
g
in the SW40 tube.
1.1.9
LDs are then collected from the top of the gradient.
Note
: RHA 1 is usually passed through the French pressure cell three
times in order to homogenize the cells efficiently.
1.2 C. elegans
(
Fig. 1.1
B,
C. elegans
)
1.2.1
4
1.1.8
The gradient is centrifuged at 256,136
10
5
worms are washed off from the NGM plates.
1.2.2
The worms are pelleted by centrifugation at 1000
g
to remove
contamination of bacteria (i.e.,
E. coli
food source).
1.2.3
Repeat steps 1.2.1 and 1.2.2 thrice.
1.2.4
The worms are washed twice with Buffer A and further
suspended in the Buffer A.
