Biology Reference
In-Depth Information
Ontario, Canada) and passed through a CH-30 Column Heater (Eppendorf, Missis-
sauga, Ontario, Canada) set at 37
C. Reaction products are monitored at 500 nm in
real time using a Programmable Detector Module (model 166, Beckman Coulter). Un-
der this setup, fractions that elute from the 22nd to 25th minute contain VLDL-sized
particles and fractions that elute from the 38th to 53th minute contain HDL-sized par-
ticles. To analyze protein contained in different fractions, we collect one fraction every
4 min (2 ml) from the 22nd to 58th minute, precipitate proteins with 2 volumes of ice-
cold acetone for 30 min at
20
C, resuspend the protein pellet in 50
l of SDS-PAGE
sample buffer, and analyze the protein content by SDS-PAGE and immunoblotting.
Several reference proteins, including Ces3/TGH, MTP, and apoE, can be used to eval-
uate successful separation of LLDs of varies size. We found that Ces3/TGH is present
in eluent from 26 to 58 min and peaks around 42 to 58 min, apoE is eluted at around
26-38 min, while MTP elutes later than apoE, at around 42-58 min.
Gradient native PAGE.
Twenty five microliter aliquots of isolated LLDs are mixed
with an equal volume of 2
native PAGE loading buffer and applied to a 2-10% gra-
dient nondenaturing polyacrylamide gel, casted with a gradient maker (Hoefer, Inc.).
Proteins are resolved in native PAGE running buffer without SDS at 80-120 V using a
BioRad Mini-PROTEAN system. Particle size is determined by comparison with the
migration of purified lipoprotein standards listed in the table below:
m
Size Category
Molecular Mass (kDa)
Diameter (nm)
HDL1
440-669
12.2-17.0
HDL2
232-440
10.5-12.2
HDL3/preb1
66-232
7.1-10.5
7.2.3
Use of BODIPY fatty acids for visualization of CLD
dynamics
(Table 7.3)
7.2.3.1
Preparation of mouse hepatocytes grown on the cover slip or
glass bottom dish
This protocol uses primary mouse hepatocytes isolated by collagenase perfusion of
the mouse liver. Details of the preparation can be found in previous publications
(
Yao & Vance, 1988
). We use fasted mice. The isolation should be performed im-
mediately before each experiment since cultured primary hepatocytes gradually lose
their metabolic properties, such as expression of some key enzymes involved in lipid
metabolism. Hepatocytes should not be older than 72 h and preferably used within
48 h after seeding (
Table 7.3
). We seed 0.2
10
6
cells for each well in a six-well
plate. For proper imaging of cells by confocal microscopy, cells should be plated
on glass cover slips with thickness compatible with the microscope objectives to
be used. If a microscope has a culture chamber or adaptor for live-cell imaging, cells
can be seeded onto a regular round cover slip placed at the bottom of a six-well plate;
