Biology Reference
In-Depth Information
3. Buffer B
(20 mM HEPES, 100 mM KCl, and 2 mM MgCl
2
, pH 7.4) is used for
washing the isolated LDs.
4. Chloroform and acetone
are used to dissolve the LD lipids and precipitate the LD
proteins.
5. SDS sample buffer
(2
, 125 mM Tris, 20% (vol/vol) 4% (wt/vol) SDS,
glycerol, 4% (vol/vol) 2-mercaptoethanol, and 0.04% (wt/vol) bromophenol
blue, pH 6.8) is used to dissolve the proteins that are subsequently separated by
SDS-PAGE.
6. Silver staining
and
Colloidal blue
staining kits are used to stain proteins in SDS-
PAGE. 100% acetonitrile, 10 mM DTT, 55 mM iodoacetamide, 25 mM
ammonium bicarbonate, 10 ng/
l trypsin are used to process the LD protein
samples before mass spectrometry analysis.
m
1.3
METHODS
We herein summarize methods that have been utilized in our previous LD proteomic
studies using bacteria,
C. elegans
, and mammalian. These studies have successfully
identified many LD proteins, especially some new LD resident proteins as well as
some new LD marker proteins (
Bartz, Zehmer, et al., 2007; Ding et al., 2012; Liu
et al., 2004; Zhang et al., 2011, 2012
). In fact, to obtain high quality LDs, not only
a general method is necessary but also developed skills to isolate and analyze LDs are
perhaps more important aspects of the technique.
Step 1
Sample preparation and LD isolation
LDs from different tissues or organisms are prepared as previously described
(
Ding et al., 2013
). The brief protocols are listed:
1.1
Bacteria (
Fig. 1.1
A, RHA 1)
Bacterium RHA 1 is cultivated aerobically in nutrient broth in
Erlenmeyer flasks at 30
C. When OD600 reaches to 2.0 40-ml culture of
cells are harvested by centrifugation and then cultivated for 24 h in 400-ml
mineral salt medium with 0.5 g/l NH
4
Cl as a nitrogen source plus 10 g/l
gluconate sodium as a carbon source.
1.1.1
400-ml liquid culture (stationary phase) is harvested via
centrifugation for 10 min at 3000
g
.
1.1.2
The cell pellet is washed twice with 30 ml PBS and once with
Buffer A.
1.1.3
The cell pellet is suspended in Buffer A.
1.1.4
The cell suspension is homogenized via the French pressure cell.
1.1.5
The sample is centrifuged at 3000
g
for 10 min to remove the
nucleus.
1.1.6
8 ml of post nuclear supernatant (PNS) is then loaded into a
SW40 tube.
1.1.7
2 ml of Buffer B is loaded on top of the sample.
