Biology Reference
In-Depth Information
FIGURE 10.3
Association of HCV and human proteins with lipid droplets. Western blot analysis of
cell extracts or isolated lipid droplet fractions (see protocol for LD isolation) from either
na¨ve Huh7.5 or HCV replicon cells (genotype 1b).
10.2.1.3
Membrane flotation assay
Yeaman et al. developed a 10-20-30% iodixanol density gradient to study protein
trafficking of the Sex6/8 protein complex in epithelial cells (
Yeaman, Grindstaff,
Wright, & Nelson, 2001
). This type of gradient results in a linear increase in density
over almost the entire length of the gradient, and membrane particles are separated in
the gradient based on their buoyant characteristics. Cells are broken mechanically in
a buffer lacking detergent to preserve membranes in the lysate. After loading the cell
lysate at the bottom of the gradient, membranes float up as the iodixanol gradient
self-generates. The separation of different membranes depends on their lipid content
and the concentration of associated proteins. After centrifugation, fractions can be
sampled from the top of the gradient and analyzed using standard SDS-PAGE
and western blot analysis for proteins of interest. We use this iodixanol density gra-
dient method to analyze membranes and proteins that are used by HCV for RNA
replication.
1.
Huh7.5 cells or HCV replicon cells (as described above) are trypsinized,
washed, and counted. A total of 3
10
7
cells for each sample are resuspended
in 3.5 ml PBS containing 0.25 M sucrose (PBS/sucrose) plus protease inhibitor
cocktail (Sigma).
2.
The cells are lysed with 200 passages in a tight-fitting Dounce homogenizer
(Wheaton) to ensure approximately 90% lysis.
3.
The cell lysate is spun at 2500
g
for 10 min at 4
C to pellet cellular debris and
nuclei. The resulting supernatant is referred to as crude lysate.
4.
Equal amounts of protein (5 mg) for each cell type are adjusted in volume to
2 ml with PBS/sucrose and mixed with 2 ml of 60% iodixanol (Sigma) resulting
in a 30% iodixanol concentration. A discontinuous iodixanol gradient (10%,
20%) is layered on top of the lysate/iodixanol mixture, and the gradient is spun
at 200,000
g
for 16 h at 4
C in a SW41T Rotor.
