Purification of Rat Liver Golgi Stacks
The study of intracellular organelles has been facilitated
greatly by their purification from cellular
homogenates. Such protocols yield an abundant
source of material for both structural and functional
studies. This article describes some simple protocols
derived from several earlier methods (Leelavathi et al.
1970; Fleischer and Fleischer, 1970; Hino et al.
, 1978) for
oµaining highly purified Golgi stacks from rat liver
and for determining their relative purity over the
II. MATERIAL AND
Centrifugation is carried out using an L8-70M or
Optima LE-80K ultracentrifuge and either a SW-28
rotor (Cat. No. 342204) containing ultraclear tubes
(Cat. No. 344058) or a SW-41 rotor (Cat. No. 331336)
containing ultraclear tubes (Cat. No. 344059) from
Beckman Coulter Inc. A 150-µm-mesh stainless-steel
laboratory test sieve (Cat. No. 200SBW.150) and a
stainless-steel receiver (Cat. No. 200BRASREC) are
from Endecotts Ltd. A 0-50% Delta refractometer (Cat.
No. 2-70) is from Bellingham and Stanley Ltd. Scintillation
counter LS6500, spectrophotometer DU530 is
from Beckman Coulter Inc.
Potassium phosphate dibasic (Cat. No. 3252), potassium
phosphate (Cat. No. 3246), sucrose (Cat. No.
4072), magnesium chloride (Cat. No. 2444), concentrated
hydrochloric acid (Cat. No. 9535), and dimethyl
sulfoxide (DMSO, Cat. No. 9224) are from J. T. Baker.
Tris (Cat. No. AB02000), SDS (Cat. No. AB01920), and
β-mercaptoethanol (Cat. No. AB01340) from American
Bioanalytical. The Bio-Rad protein assay (Cat. No. 500-
0006) is from Bio-Rad. Manganese chloride (Cat. No.
M-3634), Triton X-100 (Cat. No. T-9284), phosphotungstic
acid (Cat. No. P-4006), ovomucoid (Cat. No.
T-2011), ATP (Cat. No. A-7699), UDP-galactose (Cat.
No. U-4500), pepstatin A (Cat. No. P-5318), and
sodium cacodylate (Cat. No. C4945) are from Sigma.
Complete EDTA-free tablet (protease inhibitor cocktail
tablets, Cat. No. 1 873 580) is from Roche. [3
with a specific activity of 30-50Ci/mmol
(Cat. No. NET758) is from NEN Research products.
Ethyl alcohol (190 proof, ACS/USP grade) is from
Pharmco products, Inc. Scintillation fluid (Opti-Fluor,
Cat. No. 6013199) is from Packard BioScience. All
reagents are of analytical grade or better. The water
used is double distilled and filtered.
A. Purification of Rat Liver Golgi Stacks
0.5M potassium phosphate buffer, pH 6.7: Make up
500-ml solutions of 0.5 M anhydrous K2HPO4 (43.55 g)
and 0.5 M anhydrous KH2PO4 (34.02 g). To 400 ml of the
latter, gradually add the former until the pH reaches
6.7. Store at 4°C.
- 2 M sucrose: Dissolve 342.3 g in water prewarmed
to 50°C. Make up to a final volume of 500 ml and store
- 2M MgCl2: Dissolve 40.7g of MgCl2·6H2O in water to a final volume of 100ml. Store at room
- Protease inhibitors: Directly dissolve one EDTAfree
tablet in 50ml solution and then add 50µl pepstatin
A (5 mM stock). To prepare the stock solution of
pepstatin A, dissolve 3.43mg of the powder in 1 ml
DMSO and store at -20°C.
- Gradient buffers: Make up buffers A-E from the
preceding three stock solutions and cold water as
shown in Table I. Fifty milliliters of each buffer A, B,
and E and 100ml of buffers C and D are needed for
a medium-scale preparation. EDTA-free protease
inhibitor tablet (1 tablet per 50 ml buffer) and pepstatin
A (5µM) are added to buffer C and D. We normally
make 500ml buffer C without protease inhibitors
(which are expensive) for washing the liver. The water
should be precooled to 4°C overnight to ensure that all
the buffers are ice cold.
It is very important to be as accurate as possible
when mixing various components and to check the
refractive index of each buffer using a refractometer.
The final refractive index should be adjusted to within
±0.5% sucrose (about 0.001 in refractive index) for
buffers C and D in particular.
1. Standard Protocol (Medium-Scale Preparation)
This common protocol is used for general biochemical
studies of Golgi membranes. It suffices for many
electron microscopy studies as well.
- Starve six female Sprague-Dawley rats (body
weight 150-200 g) for 24 h.
- After killing the rats with CO2, rapidly remove
the livers into large volume of ice-cold buffer C
without protease inhibitors. This cools the liver and
washes off the blood. Then transfer the livers into icecold
buffer C with protease inhibitors. Mince into small pieces (approximately 4-5 mm in diameter) with
a pair of scissors (Fig. 1A). Put the livers into a 50-ml
- Check the weight of livers, which is normally
40-45g. A maximum of 50g liver can be used for 12
- Homogenize the tissue by gently pressing
through a 150-µm-mesh stainless-steel sieve with the
bottom of a 250-ml conical flask using a rolling action
(Fig. 1B). Adding a small amount of buffer C (with
protease inhibitor) to the sieve will make it easier to
press the liver through the mesh (Fig. 1C). Pool the
homogenate (Fig. 1D) into a 50-ml Falcon tube; the
final volume should be about 50 ml. Keep 200 µl on ice
for enzyme assay.
- Gradients (Fig. 2A):
- Place 6 ml of buffer D in each of the 12 SW-41
ultraclear tubes (size 14 × 89mm).
- Overlay 4.5 ml of homogenate.
- Overlay 1.8ml of buffer B and balance the
tubes to within 0.01 g.
- Centrifuge at 29,000rpm in a SW-41 rotor for
60min at 4°C.
- Aspirate and discard the lipid at the top and the
cytosol (colored red by hemoglobin) and collect Golgi
fractions that accumulate at the 0.5/0.86M interface
(cloudy band) with a plastic Pasteur pipette (Fig. 2B).
- Pool the fractions in a 50-ml Falcon tube. Typically,
the total volume is about 15 ml with a refractive
index of about 1.370 (0.77M sucrose). Keep 200B1 on
ice for enzyme assay. Adjust the Golgi sample to
0.25M sucrose (refractive index 1.345) using buffer A.
Make up to 50 ml with buffer B if necessary.
- For the second gradient (Fig. 2C), put 1 ml of
buffer E followed by 2ml of buffer C into each tube
and then overlay 8.0ml of the diluted Golgi fractions.
- Centrifuge at 8000rpm in a SW-41 rotor for
30min at 4°C.
- Aspirate and discard the supernatant and
collect the membranes (thin band) at the 0.5M/1.3M sucrose interface (Fig. 2D). Gently mix the Golgi membranes
(about 1.5-2ml) with 1 volume buffer A. Final
volume is 3-4ml. Check the protein concentration
by the Bradford assay (Bio-Rad); typically this is 1-
- Aliquot and freeze samples in liquid nitrogen
and then store at -80°C. Samples can be thawed and frozen twice without significant loss of enzymatic
activity or loss of morphology.
|FIGURE 1 Steps in homogenizing rat liver. (A) Mince rat liver in ice-cold buffer C. (B and C) Gently press
the liver through the metal mesh. (D) Homogenate in the sieve receiver.
2. Large-Scale Preparation
|FIGURE 2 Sucrose gradients for Golgi purification from rat liver. (A) Liver homogenate was first loaded
onto a 0.86M sucrose cushion (buffer D) and overlaid with 0.25 M sucrose (buffer B). Letters on the left indicate
sucrose buffers used to make the gradients. (B) After ultracentrifugation, Golgi stacks are enriched at the
0.5/0.86M interface. (C) Crude Golgi sample from the first gradient was loaded onto the second gradient.
(D) After centrifugation, Golgi stacks form a thin band at the 0.5/1.3M interface and can be collected using
a Pasteur pipette.
This protocol ensures large amounts of Golgi membranes
with minimal contamination by other membranes.
However, stacked cisternal structures may be less well preserved. The addition of protease inhibitors
during the procedure is optional (see Section IV). Two
consecutive spins at high speed are used to accommodate
the large amount of homogenate used.
B. Determination of β-1,4-
- Use 8-12 female rats depending on size and
requirement. Starve for 24h. Kill the rats and ensure
that all subsequent steps are performed as quickly as
- Rapidly remove the livers into 250ml of ice-cold
buffer C (without protease inhibitors) while swirling
to speed cooling.
- Weigh the livers, transfer to fresh buffer C, and
mince the tissue into small pieces (about 5mm in
diameter) with a pair of scissors (Fig. 1A).
- Homogenize the tissue by gently pressing
through a 150-µm-mesh stainless-steel sieve using the
base of a 250-ml conical flask (Figs. 1B and 1C). Take
100 µl of the homogenate for assay and record the total
volume. The final volume for up to 100g of liver
should be about 120ml, which is the maximum for
- Place 16 ml buffer D in each of the six SW-28
- Overlay each gradient with 18-20ml of the
- Finally overlay with buffer B to top up and
balance the tubes.
- Centrifuge at 28,000rpm in a SW-28 rotor for
60min at 4°C.
- Aspirate the lipid and collect Golgi fractions that
accumulate at the 0.5/0.86 M interface between buffers
C and D. About 3-4 ml should be collected from each
- Pool the fractions and dilute to 0.5M sucrose
using buffer A. Check the refractive index: 0.5M sucrose reads 1.357. Record the volume and keep
100µl for enzyme assay. The total volume should be
about 40ml and should not exceed 60ml.
- For the second gradient, add 2ml buffer E into
SW-41 tubes and overlay 8-10 ml diluted Golgi sample.
Spin at 8000rpm for 30min at 4°C.
- Aspirate and discard supernatant as before.
Collect membranes at the 0.5/1.3M sucrose interface.
Gently mix the Golgi sample (about 2.5ml)
with 1 volume buffer A. Final volume is about 5 ml.
Check the protein concentration by the Bradford
assay (Bio-Rad), which should typically be about
- Aliquot and freeze samples in liquid nitrogen
and store at -80°C.
The relative purification of the Golgi stacks can be
assessed by measuring the increase in specific activity
of the Golgi enzyme β-l,4-galactosyltransferase (GAIT)
over that of the whole liver homogenate. The enzyme
assay used here is that of Bretz and Staubli (1977),
which measures the addition of tritiated galactose
onto the oligosaccharides of an acceptor protein,
- 0.4M sodium cacodylate, pH 6.6: Dissolve 17.1g
in 150ml of water and adjust the pH to 6.6 with
HCl. Make up to 200ml and store at room
- 175mg/ml ovomucoid: Dissolve 1 g in water and
make up to a final volume of 5.7 ml. Filter through
a 0.45-µm nitrocellulose filter, aliquot, and store at
- 10mM UDP-galactose: Dissolve 25mg in a final
volume of 4.4ml of water. Aliquot and store at
- 10% (w/v) Triton X-100: Dissolve 10 g in 80 ml of
water and make up to 100ml. Store at 4°C.
- 0.2 M ATP: Dissolve 605 mg in 3 ml of water. Adjust
the pH to 6.5-7.0 with 1M NaOH and make up to
a final volume of 5ml with water. Aliquot and
store at -20°C.
- 2 M MnCl2: Dissolve 9.9 g of MnCl2·4H2O in 15 ml
of water and make up to 25 ml. Store at 4°C.
- 1% phosphotungstic acid/0.5M HCl (PTA/HCl):
Dissolve 5g of phosphotungstic acid in 400ml of
water. Add 22ml of concentrated HCl and make
up to 500ml with water. Store at 4°C.
- 5% (w/v) SDS: Dissolve 5g of SDS in 80ml of water
and make up to 100ml. Store at 4°C.
- 2M Tris: Dissolve 24.2 g of Tris in 70ml of water
and make up to 100 ml. Store at room temperature.
- Assay mixture: Make up a fresh batch of the
assay mixture from the aforementioned stocks as
follows: 200µl sodium cacodylate, 6µl β-mercaptoethanol,
200µl ovomucoid, 40µl UDP-galactose,
40 µl Triton X-100, 20 µl ATP, 40 µl MnCl2, 10 µl
[3H]UDP-galactose, and 1040 µl of water. The concentration
of UDP-galactose in the assay mixture
is 0.25 mM.
C. Determination of Protein Concentration
- Once the Golgi preparation has been completed,
make 1:20 dilutions of the homogenate, intermediate,
and Golgi fractions using water.
- Add 80µl of assay mixture (in duplicate) to screwcapped
Eppendorf tubes containing 20µl of the
diluted samples or water (blanks). Vortex and incubate
at 37°C for 30min.
- Stop the reaction by adding 1 ml of ice-cold
PTA/HCl and spin at 13,000rpm on a bench-top
centrifuge for 10s.
- Aspirate and discard the supernatants. Add 1 ml of
PTA/HCl. Resuspend the pellets by vortexing and
spin as in step 3.
- Aspirate and discard the supernatants. Add 1 ml
of ice-cold 95% ethanol and resuspend the pellets
as in step 4.
- Spin as in step 3 and discard the supernatant.
Resuspend the pellets in 50µl of 2M Tris followed
by 200 µl of 5% SDS. Shake or vortex until dissolved.
- Add 10µl of assay mixture (containing 2.5 nmol of
UDP-galactose), 40 µl of water, and 200 µl of 5% SDS
to a fresh tube. This allows determination of the
[3H]UDP-galactose-specific activity in the mixture.
- Add 1 ml of scintillation fluid to each sample. Vortex
and count in a scintillation counter using the tritium
D. Calculations of Purification Tables
- While the GaIT assays are incubating, make up the
following dilutions of the three samples with water:
1:100 for the homogenate, 1:20 for the intermediate
fraction, and 1:2 for the Golgi fraction.
- Prepare a diluted Bio-Rad protein assay solution
(fivefold dilution with water)
- Aliquot 10 µl of each of the diluted samples in duplicate
into 1-ml cuvettes using 10µl water as a blank.
Also aliquot 10µl of 0.1, 0.2, 0.4, and 0.8mg/ml of
bovine serum albumin (BSA) for preparation of a
- Add 990µl of the diluted Bio-Rad protein assay
solution, mix well, and measure the absorbance at
- Construct a protein standard curve by plotting the
absorbance of each standard against the concentration
of BSA. Calculate the slope (m) and the intercept
at the ordinate axis (c).
|Protein concentration (mg/ml) =
||(sample absorbance- c) × dilution
Total protein (mg) = Protein concentration (mg/ml) × volume (ml)
- Calculate the specific activity (SA) of [3H]UDPgalactose
in the assay mixture as
|SA[3H]UDP-galactose (dpm/nmol) =
||dpm of standard- blank
10 µl assay mixture contains 2.5 nmol of UDP-galactose
(Section III, B, step 7).
- Calculate the GalT activity in each sample as
|GalT activity concentration (nmol/h/ml) =
||average dpm- blank
||× 20 (dilution factor)
Total GaIT activity (nmol/h) = GaIT activity
concentration (nmol/h/ml) × volume (ml)
- Calculate the SA of GalT by dividing its concentration
by the protein concentration of the same
sample to give SA in nanomoles per hour per milligram
- The yields of Golgi membranes can be calculated
from the ratio between the total GalT activity in the
Golgi fractions and that of the homogenate.
- The purification fold is the factor by which the SA
of GalT increases in the Golgi fractions over the
This protocol typically yields Golgi membranes
that are purified 80- to 100-fold over the homogenate,
as depicted in Table II. The Golgi preparations
contain very little lysosomal or endoplasmic reticulum
contamination as assessed by assay of β-N
(Landegren, 1984) and
rotenone-insensitive NADH-cytochrome c reductase
(Sottocasa et al.
, 1967). The stacked nature of these
Golgi membranes can be confirmed by examination of
preparations by electron microscopy (Fig. 3). Samples are fixed in suspension by the method described by
Pypaert et al.
The addition of protease inhibitors, although not
essential, is recommended, especially when the Golgi
protein of interest is known to be sensitive to proteases.
The addition of protease inhibitors can reduce
the apparent purification factor, although the reasons
are not clear.
|FIGURE 3 Representative micrographs of a typical rat liver Golgi preparation showing stacked membranes
at low (A) and high (B) magnification. Bars: 1 µm (A) and 0.2µm (B).
- As with many organelle purification procedures,
it is vital to keep all solutions at 4°C during the whole
protocol to prevent excessive protease digestion. If
possible, steps 3-5 should be performed in a cold room. The addition of a protease inhibitors helps limit
- All steps should be carried out as quickly as possible,
and the entire procedure, from killing of the rats
to freezing of the final samples, should take approximately
- Gradients should not be overloaded by increasing
the concentration of the homogenate, as this
increases the amount of mitochondrial contamination.
- The final Golgi pellet should be white. A brown
pellet indicates the presence of contaminating
mitochondria. Lowering the concentration of the
homogenate reduces such contamination.
- The 150-µm sieve will become clogged with connective
tissue after excessive use. This can be removed
after each preparation by soaking the sieve in 4M NaOH for 20-30min followed by washing with copious amounts of water and brushing. Prolonged
soaking in 4M NaOH attacks the glue used in binding
the mesh to the wall of the sieves and should therefore
The authors thank the former and current members
of the Warren laboratory for contributions to the development
of these methods, Henry Tan for providing the
photographs in Fig. 1 and 2, and Matthew Beard for
critical reading of the manuscript.
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