Biology Reference
In-Depth Information
Known CLD proteins should be immunoblotted as controls using specific antibodies.
For this purpose, perilipin 2 is an excellent marker for CLD recovery in the liver. In
order to determine the purity of the preparation, cytosolic proteins and resident poly-
topic membrane proteins from other organelles such as the ER and mitochondria
should be analyzed by immunoblotting with specific antibodies. Polytopic ER mem-
brane proteins (such as phosphatidylethanolamine-
N
-methyltransferase) are good
markers for the absence of ER contaminations from CLDs. Likewise, fumarase, with
mitochondrial and cytosolic localizations, is a reliable marker for contamination of
CLDs with mitochondrial components.
7.2.1.3
Solubilization of CLD-associated proteins for immunoblotting
In order to solubilize CLD-associated proteins, fresh CLD fractions should bemixedwith
10%SDS (1:1, v/v) and incubated for 1-2 h at 37
C in a sonicating water bath with con-
stant agitation. Then, samples should be centrifuged in a microcentrifuge for 10 min at
maximum speed at room temperature and the infranatants containing the solubilized pro-
teins should be collected from beneath the floating lipid layer. For this purpose, it is very
helpful to use a 200
l tip inserted very carefully through the floating fat cake all the way
down to the bottom of the tube. It is important not to disrupt the lipid cake; should this
happen, recentrifugation should be performed. Transfer the infranatant to a 1.5 ml tube
and add equivalent volumes of 2
m
SDS electrophoresis sample buffer. Then, boil sam-
ples for 10 min and finally load them onto a discontinuous SDS-PAGE gel.
7.2.2
Isolation of LLDs from mouse liver
7.2.2.1
Preparation of the mouse
To prepare mice for LLD isolation, we fast mice overnight (12-16 h) before exper-
iments because under fasting conditions, large amounts of LDs are accumulated in
the liver. Instead of simply removing food from the mice, fasting should be done by
transferring mice into a clean cage with free access to water. Usually up to four livers
are used for an experiment. If the study requires comparing different feeding condi-
tions, then much more material is required. The following method is performed using
fasted mice.
7.2.2.2
Isolation of the liver and tissue homogenization
Anesthetize the mice to a state suitable for surgery by inhaling of metophane or iso-
flurane. Vital signs should be monitored during this procedure. Exsanguinate the
mice via cardiac puncture and carefully remove the liver, which is immediately
transferred to ice-cold TBS (
Table 7.2
). After rinsing with TBS, livers are weighed
and homogenization buffer is added to make a final 20% (w/v) homogenate. For
homogenization, we use a motor-driven Potter-Elvehjem homogenizer (Wheaton
Science Products, Millville, NJ) at medium speed (levels 3-4) for 10 strokes in ho-
mogenization buffer. Proper homogenization is one of the most important steps for
the preparation in order to efficiently break cells while maintaining the integrity of
subcellular content. Alternative devices such as Polytron or loose-fitting Dounce
