Biology Reference
In-Depth Information
important step to remove proteins and CLDs peripherally associated with the micro-
somal membrane. Microsomal membranes are pelleted by ultracentrifugation at
106,000
g
for 1 h, while peripheral proteins (cytosolic side)
remain in the
supernatant.
7.2.2.4
Release of microsomal lumenal content
To release microsomal lumenal contents, pelleted microsomes are resuspended in
1 mM Tris-HCl (pH 8.8) in a volume equivalent to 1/3 of the original homogeniza-
tion buffer and incubate on ice for 30 min. This step swells microsomes by osmotic
pressure and preserves the integrity of LLDs and the associated proteins. At the end
of the incubation, the swelled microsomes are disrupted by homogenization with
Potter by three strokes at low setting to release lumenal contents. The mixture is then
centrifuged at 106,000
g
for 1 h to obtain lumenal contents (supernatant) and
microsomal membranes (pellet).
7.2.2.5
Separation of LLDs from VLDL precursors
At least two types of LDs are present in the microsomal lumen: the apoB-containing
particles (including mature VLDL and VLDL precursors) and the apoB-free LLDs.
To remove apoB-containing particles from LLDs, the microsomal lumenal contents
obtained from the previous step are adjusted to 50 mM Tris-HCl, 150 mM NaCl
(pH 7.4), and apoB-containing particles are immunoprecipitated using anti-apoB
polyclonal antibodies (goat anti-apoB from Chemicon). It is important that the im-
munoprecipitation is performed in the absence of detergents in order to preserve the
integrity of LLDs. Two forms of apoB are present in the rodent liver: apoB48
(
500 kDa). The antibodies we use recognize both forms
of apoB. For each milliliter of lumenal content, usually 5
250 kDa) and apoB100 (
l of antibodies are added
and the mixture is incubated while rotating the tubes end-over-end at 4
C overnight.
Then 20
m
l protein A Sepharose beads prewashed with PBS are added and incubated
with the mixture end-over-end at 4
C for 2 h to form protein-bead complex, which
is subsequently pelleted by centrifugation for 30 s at 6000
m
g
. The IP is then washed
3
with PBS and prepared for SDS-PAGE and immunoblotting. The supernatant
(post-IP) is collected for further purification, which is described below.
7.2.2.6
Isolation of microsomal LLDs by density gradient
ultracentrifugation
Two milliliters of post-IP supernatant are combined with an equal volume of glycerol
and transferred to a transparent ultracentrifuge tube suitable for SW40 swing bucket
rotor. We prefer the Beckman Ultra-Clear
TM
centrifuge tube, since it allows for good
inspection of the gradient set up. The glycerol-adjusted samples are then overlayed
with 4 ml of homogenization buffer and an additional layer of 4 ml of TBS. The gra-
dient should be set with great care. We find using a long needle of 18G or narrower is
helpful to gently deliver overlay solutions without disturbing the lower layer. Once the
gradient is set, the samples are centrifuged in a Beckman SW40 rotor at 35,000 rpm
(160,000
g
) for 2 h at 8
C. Be sure to use a swinging bucket and avoid fixed angle
