Biology Reference
In-Depth Information
emerging LD proteomics. These proteomic studies have spanned multiple species
from bacteria to humans and demonstrated that LDs have quite different protein
compositions, suggesting that the function of LDs varies between organisms, but
also the regulation. However, more research needs to be conducted as only limited
information is currently available, including further LD isolation and proteomic
studies so as to fully understand the organelle. We have successfully isolated LDs
and carried out proteomic analyses from a bacterium
Rhodococcus
RHA 1 (
Ding
et al., 2012
), a nematode
Caenorhabditis elegans
(
Zhang et al., 2012
), a mamma-
lian cell line CHO K2 (
Bartz, Zehmer, et al., 2007; Liu et al., 2004
), and mam-
malian tissues, that is, mouse liver (
Bartz,Li,etal.,2007
) and skeletal muscle
(
Zhang et al., 2011
). Herein we provide a detailed experimental procedure that
summarizes and standardizes LD proteomic studies from these three representa-
tive species. We believe that the established method sufficiently allows the iso-
lation of relative purer LDs (
Ding et al., 2013
) and proteomic analysis
theoretically from all species mentioned.
1.1
EQUIPMENT
1. French press cell
(JNBIO, cat. no. JN-3000, high-pressure homogenizer) is used
for homogenization of bacteria such as
Rhodococcus
sp. RHA 1.
2. Polytron homogenizer
(Cole-Parmer, LabGEN 700 homogenizer) is used to
disrupt the cells of
C. elegans
.
3. Dounce homogenizer
(Wheaton, cat. no. 357542) is used to dissect the cells of
mouse skeletal muscle and liver.
4. Nitrogen bomb
(PARR Instrument, 4639 cell disruption bomb) is used to
homogenize tissue culture cells,
C. elegans
, and mouse skeletal muscle and liver.
5. Ultracentrifuge
(Beckman, Optima
L-100 XP ultracentrifuge) and SW40 Ti
rotor are used to separate LDs from other cellular fractions.
6. Ultracentrifuge
(Beckman Coulter, Optima MAX ultracentrifuge) and TLA100.3
rotor are used to obtain cytosol (Cyto) fraction.
7. Particle analyzer
(Beckman Coulter, Delsa
™
Nano C particle analyzer) is used
to analyze LD size.
8. Confocal microscope
(Olympus, FV1000) and
transmission electron microscope
(FEI, Tecnai 20) are used for morphological study of LDs.
9. Nano-LC-ESI-LTQ mass spectrometer
and
nano-LC-ESI-LTQ Orbitrap XL mass
spectrometer
are used for the proteomic analysis of different LD samples.
™
1.2
REAGENTS
1. Phosphate buffered saline
(
PBS
) is used to clean samples.
2. Buffer A
(20 mM tricine and 250 mM sucrose, pH 7.8) is used to protect
intracellular organelles in the sample homogenization.
