Materials and Methods
Preparation of Membrane Fractions Suitable for 2D electrophoresis
Three Male BALB/c mice, age 6-7 weeks from HSLAS, University of Alberta, provided
with standard diet and water, were killed by cervical dislocation. Livers (2-3 g) were removed
quickly and minced with a razor blade. Subsequent steps performed on ice. Minced livers were
placed in 6 ml of homogenization buffer (37.5 mM Tris-maleate, pH 6.4; 0.5 M sucrose; 1%
dextran and 5 mM MgCl2). Homogenized with a Polytron for 40 seconds at setting 4 and then
centrifuged at 5,000g (6,000 rpm in JA-17 rotor) for 15 minutes. The supernatant was used for
endoplasmic reticulum isolation, the top 1/3 of pellet was used for golgi isolation and the lower
½ of pellet was used for plasma membrane isolation. Sucrose solutions were prepared in 37.5
mM Tris-maleate, pH 6.4; 1% dextran; and 5 mM MgCl2 except for the discontinuous gradient
used for the isolation of plasma membrane in which the sucrose gradients are prepared in
distilled, de-ionized water. All centrifugation steps were performed at 4 C.
ER isolation
After the initial centrifugation at 5,000g for 15 minutes at 4 C, the supernatant was taken
and diluted 1:4 (add 18 ml) with homogenization buffer (37.5 mM Tris-maleate, pH 6.4; 0.5 M
sucrose; 1% dextran and 5 mM MgCl2) and centrifuged at 8500g (7250 rpm JA17 rotor) for 5
minutes to remove contaminating mitochondria. The pellet was saved for subsequent
mitochondrial purification (see later). The endoplasmic reticulum containing supernatant (24 ml)
was top loaded onto a discontinuous sucrose gradient consisting of 6 ml 2.0 M sucrose, 8 ml 1.5
M sucrose and 8 ml of 1.3 M sucrose in a polyallomer centrifuge tube (25 x 89 mm). The
gradient was then centrifuged at 90,000g in a swinging bucket rotor (25,000 rpm in SW27 rotor)
for 120 minutes. The endoplasmic reticulum fractions were collected at the 1.3 M – 1.5 M
interface and the 1.5 M – 2.0 M interface. The pooled fractions were diluted 1:3 with 10 mM
Tris-HCl, pH 7.5 and centrifuged at 90,000g (25,000 rpm in SW27 rotor) for 45 minutes. The
pellet was resuspended in 10 mM Tris-HCl, pH 7.5 again and centrifuged at 90,000g for 45
minutes. The pellet was then resuspended in 2 ml 10 mM Tris-HCl, pH 7.5 and aliquoted into 10
small (T100 rotor) centrifuge tubes and then centrifuged at 63,000 rpm (T100 rotor in Beckman
bench top ultracentrifuge) for 30 minutes. The supernatant was removed and the pellets were
subsequently frozen in the –80 C freezer for loading onto the 2D system.
Golgi Isolation
The yellow/brown portion or upper 1/3 of the initial pellet was resuspended in a small
part of supernatant with a large bore Pasteur pipet and transferred to a new tube. The resulting
mixture was passed through the pipet approximately 12 times to resuspend thoroughly. This
mixture was layered over 2.7 ml of 1.2 M sucrose in an ultra-clear centrifuge tube (13 x 51 mm)
and centrifuged at 100,000g in a swinging bucket rotor (30,000 rpm in SW60 rotor). Golgi
apparatus were collected from interface with care not to remove any of the 1.2 M sucrose layer
which contained ER fragments. The collected golgi was diluted in 10 mM Tris-HCl, pH 7.5 and
concentrated by centrifugation at 5500g (6500 rpm in JA17 rotor) for 20 minutes. The golgi
pellet was washed once again with 10 mM Tris-HCl, pH 7.5 and then aliquoted in 200 ul aliquots
and centrifuge in bench top centrifuge at 14 000 rpm for 25 minutes. The supernatant was
removed and the pellets were frozen at –80 C.
Plasma Membrane Isolation
The lower ½ of pellet was resuspended in 2.5 ml of 1 mM Na2HCO3 and homogenized at
lowest setting (setting 1) of polytron until homogenous. Diluted to 10 ml with 1 mM Na2HCO3
and centrifuged at 4500g (5500 rpm in JA17 rotor) for 10 minutes. The top 1/3 of the pellet was
resuspended in 2-3 ml of supernatant. This was mixed drop wise with 10 ml of 81% sucrose
(dissolved in H2O) and placed in a polyallomer centrifuge tube (25 x 89 mm). This mixture
formed the bottom layer of the sucrose gradient. On top of this was layered 3.5 ml each of 53.4
(d=1.2), 48 (d=1.18), 46 (d=1.173), 44 (d=1.166), 42.6 (d=1.16) and 40 (d=1.156) gm sucrose
per 100 ml distilled water. This gradient was centrifuged for 90 minutes at 90,000g using a
swinging bucket rotor (25,000 rpm, SW27 rotor). Plasma membranes were collected form the 40
– 42.6% sucrose interface, diluted with 10 mM Tris-HCl, pH 7.5 and collected by centrifugation
for 20 minutes (7750 rpm in JA17 rotor). The pellet was washed once again with 10 mM TrisHCl, pH 7.5 and resuspended in 800 ul of 10 mM Tris-HCl, pH 7.5 and aliquoted into 200 ul
fractions and centrifuged at 14000 rpm in bench top centrifuge for 25 minutes. The supernatant
was removed and the pellet was frozen at –80C.
Nuclei Isolation
The pellet obtained after the plasma membrane sucrose gradient contains the crude nuclei
fraction. The pellet was resuspended in 20 ml of 10 mM Tris-HCl, pH 7.5 and centrifuged at
7750 rpm in JA17 rotor for 20 minutes. This was repeated and the final pellet was resuspended
in 2 ml of 10 mM Tris-HCl, pH 7.5 and aliquoted into 200 ul fractions and centrifuged at 14,000
rpm for 25 minutes in the bench top centrifuge. The supernatant was removed and the pellets
were frozen at –80C.
Mitochondria Isolation
The crude mitochondrial fraction was found in the pellet from the first ER centrifugation.
The pellet was resuspended in 20 ml of 10 mM Tris-HCl, pH 7.5 and centrifuged at 7750 rpm for
25 minutes (JA17 rotor). This was repeated and the final pellet was resuspended in 2 ml of 10
mM Tris-HCl, pH 7.5 and aliquoted into 200 ul fractions and centrifuged at 14,000 rpm for 25
minutes in the bench top centrifuge. The supernatant was removed and the pellets were frozen at
–80C. If further purification of mitochondria is needed, the fraction can be loaded onto a sucrose
gradient and the purified mitochondrial fraction can be collected at an interface.