Isolation of Rough and Smooth
Membrane Domains of the Endoplasmic
Reticulum from Rat Liver
Subcellular fractionation from tissue homogenates
have contributed extensively to our understanding of
organelle structure and function. This article provides
details of a tissue fractionation protocol evolved
from several earlier methods (Paiement et al.
Paiement and Bergeron, 1983; Lavoie et al.
, 1996), for
the purification of rough microsomes, corresponding
to derivatives of the rough endoplasmic reticulum,
and for the purification of smooth microsomes, corresponding
to derivatives of the smooth endoplasmic
reticulum. The latter constitute a subcompartment of
the transitional endoplasmic reticulum of hepatocytes
(Lavoie et al.
, 1996, 1999; Roy et al.
II. MATERIALS AND
Sucrose solutions (0.25, 0.86, 1.0, 1.38, and 2.0M
Densities of sucrose stock solutions should be checked for
proper density with a refractometer.
Sucrose-Imidazole (0.25 mM
sucrose, 3 mM
Lint-free tissues (e.g., Kimwipes)
Potter-Elvehjem tissue grinder with ribbed Teflon
pestle (50-ml capacity)
Nylon mesh (150µm mesh, Thompson B & SH Co.
Ltd., 8148 Devonshire, Montreal, Canada)
Glass funnel (60° funnel angle, 100mm diameter)
100-ml graduated cylinder
Refrigerated low-speed centrifuge with fixed angle
rotor (e.g., Beckman-Coulter Avanti J-20 with JA
25.50 rotor or equivalent)
Loose-fitting Dounce homogenizer (7- and 15-ml
capacity, with Wheaton type B piston, at least two
Graduated conical tube
2.0-ml glass syringes with blunt-end syringe needles
(10 cm long, 2 mm diameter)
Ultracentrifuge with swinging bucket and fixed-angle
rotors (e.g., Beckman L-70 with SW60 and Ti50
Rats must be fasting for 12 to 24h prior to
- Use two male rats of 300- to 400-g body weight
(one liver weighs approximately 9 g).
- Sacrifice rats via decapitation 2 and excise livers
(free of connective tissue) and place in petri dish containing
ice-cold 0.25M sucrose.
2 Animal sacrifice must be carried out according to the "Guide to
the Care and Use of Experimental Animals of the Canadian Council
on Animal Care" or equivalent protocol required by the country
- Blot the livers using lint-free tissues and transfer
to a preweighed beaker containing ice-cold 0.25M sucrose and weigh (weight of two livers together
should be a minimum of 18 g).
- Blot the livers using lint-free tissues and chop
them finely with scissors. Allow the pieces to fall into
the mortar of the tissue grinder. 3
3 Steps 5 to 8 are carried out in a cold room.
- Add a volume of ice-cold 0.25M sucrose, which
is equivalent to 2.5 times the weight of the livers (e.g.,
18.0 g × 2.5 = 45.0 ml).
- Homogenize using the ribbed Teflon pestle for
20 strokes (down and up is one stroke) at 2850rpm
(mortar should be kept in plastic beaker filled with ice
throughout homogenization process).
- Filter the homogenate through the nylon mesh
folded over four times in the glass funnel. 4
4 The homogenate should not be squeezed through the nylon
mesh, but instead stirred gently with a glass rod to facilitate the
homogenate passing through the mesh.
- The total homogenate at this point should be
approximately 54ml (45ml sucrose plus about 9ml
liver tissue) and should be separated into 4 × 15ml
- Centrifuge in a Beckman-Coulter JA 25.50 fixed
angle rotor that is precooled to 4°C at 8700g for 13 min
to sediment cellular debris and nuclei.
- Transfer the supernatant [low-speed supernatant
(LSS fraction), see Fig. 1] into three polycarbonate
screw top tubes (10-ml capacity).
- Centrifuge in a Ti50 fixed angle rotor at
43,000g for 6 min 40 s [press the stop button of the centrifuge
at exactly 6min 40s. Complete stop (with
brakes) should occur by about 10 min 20 s after the start
- With a Pasteur pipette, recover the supernatant
[intermediate-speed supernatant (ISS fraction), see Fig.
1] and a portion of the flocculent layer that is covering
the pellet (ML fraction, containing lysosomes and
- Centrifuge the ISS fraction in a Ti50 rotor at
110,000 g for 60 min.
- Discard supernatant (S fraction, see Fig. 1).
- Resuspend the pellets in 1.38 M sucrose. 1 Recovery
of total microsomes (TM) in the pellets is done as
- Add 0.5ml of 2.0M sucrose1 to the first centrifuge
tube and resuspend the microsomal
pellet using a blunt-ended glass rod.
- Transfer the suspension to subsequent tubes,
resuspending the microsomal pellet with the
glass rod in each one before transferring on
to the next.
- After resuspension is complete, rinse all centrifuge
tubes with 2.0M sucrose and transfer
to a 7-ml Dounce.
- Repeat rinsing as needed. However, do not
exceed an accumulation of 5ml in the
- Gently homogenize the resuspensions in the
Dounce using the piston.
- Measure volume using a graduated conical
- Rinse both the centrifugation tubes and the
Dounce with 1 ml 2.0M sucrose, 1 which is transferred
to the conical tube and mixed in gently by covering the
top of the tube with parafilm and inverting the tube
two or three times. Repeat once.
- Use a refractometer to adjust the density to
1.38M by adding 0.25 or 2.0M sucrose1 to the TM
resuspension as needed.
- Bring the volume of the TM resuspension to
9.8 ml using the 1.38 M sucrose1 stock.
- Prepare six sucrose gradients using 4.0-ml tubes
destined for the Beckman SW60 rotor as follows.
- Carefully place 0.6ml of 0.25M sucrose1 in
the bottom of a Beckman SW60 tube with a
glass syringe and blunt-ended needle.
- Using a new syringe and needle, gently place
the needle at the bottom of the tube and very
gently inject 0.8 ml of 0.86M sucrose1 underneath
- Place a third syringe and needle carefully at
the bottom of the tube and inject 0.8ml of 1.0M sucrose1 very gently underneath the
previous two layers.
- Using a fourth new syringe and needle, very
gently insert needle to the bottom of the tube
again and very carefully inject 1.4ml of TM
fraction to produce the bottom layer of the
gradient (see Fig. 1).
- Repeat steps a-e for all six tubes.
- Centrifuge gradients in a SW60 swingingbucket
rotor at 53,000rpm (300,000g) for 60min.
- For smooth microsomes (SM):
- Carefully extract the top half of the bottom
step (see step 4 in Fig. 1) using a Pasteur
pipette and place in a Ti50 centrifuge tube.
- Add 0.25M sucrose-imidazole until tube is
filled and mix gently.
- For rough microsomes (RM):
- Discard all gradient steps in all tubes.
- Turn tubes upside down on a lint-free tissue
for approximately 30s to allow residual
supernatant to drip out.
- Wipe edges of tube (above pellet) using a
lint-free tissue wrapped around a glass
- Resuspend pellets using a 15.0-ml Dounce
and rinse tubes with sucrose-imidazole
(using the same sequential rinsing method as
described in Steps 16a to 16e). Transfer resuspended
rough microsomes to a Ti50 tube.
- Once sequential rinsing is done, add
sucrose-imidazole to tube so that the final
volume of sucrose-imidazole added is equivalent
to approximately 11 ml.
- Divide both SM and RM separately into two
equal portions in Beckman Ti50 tubes and centrifuge
at 140,000g for 60 min.
- Discard supernatants and resuspend as follow:
- Resuspend the rough microsomal pellet in
2.0 ml sucrose-imidazole.
- Resuspend the smooth microsomal pellet in
3.0 ml sucrose-imidazole.
- Aliquot microsomal fractions into 60-, 110-, and
210-µl portions and store at -80°C in hermetically
sealed tubes until needed.
The "rough" nature of the derivatives of the rough
endoplasmic reticulum can be confirmed by examination
of preparations by electron microscopy (Fig. 2).
Samples were fixed in suspension and processed as described previously (Paiement et al.
, 1980). Classical
rough microsomes are shown in Fig. 2A and contain
greater than 80% rough vesicles as determined by
quantification of the number of vesicles with associated
ribosomes. In Fig. 2B the microsomal fraction
consists of approximately 60% smooth microsomal
vesicles and 40% rough microsomal vesicles, which
have few (one to four) ribosomes associated with their
limiting membranes. Both microsomal fractions were
shown previously to be enriched in enzyme markers
for the endoplasmic reticulum and reduced in enzyme
markers for Golgi and plasma membrane derivatives
(Paiement et al.
, 1980; Paiement and Bergeron, 1983;
Lavoie et al.
, 1996, 1999; Roy et al.
This procedure takes approximately 12h. All
samples should be kept on ice at all times throughout this protocol. All centrifugations should take place at
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