Isolation of Nucleoli
The human nucleolus is a prominent nuclear substructure
assembled around tandemly repeated ribosomal
genes (rDNA genes) on chromosomes 13, 14, 15,
21, and 22 and is the site of rDNA transcription and
ribosome subunit synthesis (reviewed by Shaw and
Jordan, 1995). Visualised under electron microscopy,
the nucleolus is separated morphologically into three
distinct substructures: fibrillar centres (FC), which are
surrounded by dense fibrillar components (DFCs), and
granular components (GC) (Shaw and Jordan, 1995).
Its high density and structural stability allow effective
purification using a straightforward procedure. Following
the initial successful attempts to purify nucleoli
from human tumour cells and rodent liver cells in
the early 1960s (e.g., Muramatsu et al.
, 1963), numerous
studies have reported on the characterization of
isolated nucleoli. Nucleoli have been purified from a
large variety of mammalian tissues, including liver,
brain (Banks and Johnson, 1973), and thyroid (Voets et al.
, 1979), and from cells of nonmammalian species
such as Xenopus (Saiga and Higashinakagawa, 1979)
and Tetrahymena (e.g., Matsuura and Higashinakagawa,
1992). The ability to isolate nucleoli in large scale
provides an excellent starting material for identifying,
purifying, and studying nucleoli and has contributed
significantly to the understanding of this nuclear
The following protocol is based on one of the earliest
published procedures for isolating nucleoli from
cultured human cells (Muramatsu et al.
, 1963). This is
a very robust procedure in which isolated nuclei are
subjected to sonication whose power is adjusted so
that nucleoli remain intact while the rest of nuclei are fragmented. Then nucleoli are isolated by spinning
through a density gradient, exploiting their high
density compared with other nuclear components.
Detailed analysis using highly sensitive mass spectrometric
techniques (Andersen et al.
, 2002) indicated that
this method was a reproducible and efficient way to
produce highly purified nucleoli.
All solutions are supplemented with Complete protease
inhibitor tablet (Roche, Cat. No. 1-873-580) at the
final concentration of 1 tablet/50ml).
Phosphate-buffered Saline (PBS)
: 10 mM
HEPES, pH 7.9, 10 mM
KCl, 1.5 mM
, 0.5 mM
: 0.25 M
sucrose and 10 mM
: 0.35 M
sucrose, 0.5 mM
: 0.88 M
Sucrose, 0.5 mM
See "Notes" on making stock sucrose solution.
- Seed HeLa cells (ATCC number: CCL-2) onto 10
× 14-cm petri dishes and culture at 37°C in 5% CO2 in Dulbecco's modified Eagle medium (DMEM)
containing 4mM L-glutamate, 4.5 mg/ml glucose, and
0.11 mg/ml sodium pyruvate (Invitrogen UK, Cat. No:
41966-029), supplemented with 100U/ml penicillin
and 100µg/ml streptomycin [1% (v/v) penicillin/
streptomycin solution, Invitrogen UK, Cat. No: 15140-
122] until >90% confluence (approximately 107 cells per dish). This number of HeLa cells consistently provides
nucleoli with excellent yield and purity. It is possible
to scale down the preparation, although the purity of
isolated nucleoli may suffer. Make sure you monitor
every step using a phase-contrast microscope (see
later). One hour before nucleolar isolation, replace
with fresh, prewarmed medium.
- Harvest cells by trpysinization. Rinse each dish
three times with prewarmed PBS and, on removal of
the last rinse, add 2ml of trypsin-EDTA solution
(Invitrogen UK, Cat. No: 25300-054) per dish. Swirl the
dishes to make sure the trypsin-EDTA is distributed
evenly and return the dishes to the incubator for about
5min. Check under a phase-contrast microscope that
all the cells are detached. Prolong incubation if needed.
Into each dish add 8 ml of prewarmed medium and
pipette up and down so that all the cells are collected
as a single-cell suspension. Pool all the harvested cells
into 2 × 50-ml Falcon tubes. For some strains of HeLa
cells, it is also possible to harvest the cells by scraping
them in 5 ml ice-cooled PBS per dish. Because scraping
may lead to an impure nucleolar preparation in some
HeLa strains, it is not recommended as the method of
- Wash three times with ice-cold PBS at 218g (1000rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) at
- After the final PBS wash, resuspend the cells in
5 ml of buffer A and incubate the cells on ice for 5 min.
Put a small drop of the cell suspension on a glass slide
and check under a phase-contrast microscope, such as
a Zeiss Axiovert 25, using a 20× objective. The cells
should be swollen, but not burst (Fig. 1). Nucleoli
of cultured mammalian cells disassemble at 37°C in
hypotonic conditions (Zatsepina et al., 1997). It is therefore
imperative to keep the cell suspension on ice
during this step.
|FIGURE 1 HeLa cells after step 4. Note the swollen cytoplasm
and prominent nucleoli. Bar: 10 µm.
- Transfer the cell suspension to a precooled 7-ml
Dounce tissue homogenizer (Wheaton Scientific
Product Cat. No: 357542). Homogenize 10 times using
a tight pestle ("A" specification: 0.0010-0.0030-in.
clearance) while keeping the homogenizer on ice. The
number of strokes needed depends on the cell type
used (see Section III). It is therefore necessary to check
the homogenized cells under a phase-contrast microscope
after every 10 strokes. Stop when >90% of the
cells are burst, leaving intact nuclei, with various
amounts of cytoplasmic material attached. In most
cases, the presence of this cytoplasmic contamination
does not affect the final purity of the isolated nucleoli
(Fig. 2). Centrifuge the homogenized cells at 218g
(1000rpm, Beckman GS-6 centrifuge, GH-3.8 rotor) for
5min at 4°C. The pellet contains enriched, but not
highly pure, nuclei.
|FIGURE 2 Step 5 and 6 of the procedure.
- Resuspend the pellet with 3 ml S1 solution (Fig.
3). The pellet should be resuspended readily by pipetting
up and down. A pellet that cannot be resuspended
contains lysed nuclei and should be discarded. Layer
the resuspended pellet over 3ml of the S2 solution.
Take care to keep the two layers cleanly separated.
Centrifuge at 1430g (2500rpm, Beckman GS-6 centrifuge,
GH-3.8 rotor) for 5 min at 4°C. This step results
in a cleaner nuclear pellet (Fig. 3). Resuspend the pellet
with 3 ml of the S2 solution by pipetting up and down.
|FIGURE 3 Step 7 of the procedure. Note the clear boundary between S1 and S2 layers before centrifugation.
(Insets) DIC images of the supernatant and pellet. Note prominent nucleoli inside
nuclei in the pellet.
Bars: 10 µm.
- Sonicate the nuclear suspension with six 10-s
bursts (with 10-s intervals between each burst) using a
Misonix XL 2020 sonicator fitted with a microtip probe
and set at power setting 5 (Fig. 4A). Check the sonicated
nuclei under a phase-contrast microscope. There
should be virtually no intact cells and nucleoli should
be readily observed as dense, refractile bodies (Fig.
4B). The optimal sonication time depends on the cell
type used. If you attempt to isolate nucleoli from a cell
type from the first time, it is necessary to check the sonicated
material under a microscope after every 10sec
of sonication. Oversonication leads to destruction of
|FIGURE 4 (A) Setup for sonication. (B) DIC image of sonicated nuclei. Note the presence of prominent
nucleoli. Bar: 10 µm.
- Layer the sonicated sample over 3 ml of the S3
solution and centrifuge at 3000g (3500rpm, Beckman
GS-6 centrifuge, GH-3.8 rotor) for 10min at 4°C (Fig.
5). The pellet contains nucleoli, whereas the supernatant
can be retained as the "nucleoplasmic fraction"
- Resuspend the nucleoli with 0.5 ml of the S2 solution,
followed by centrifugation at 1430g (2500rpm,
Beckman GS-6 centrifuge, GH-3.8 rotor) for 5min at
4°C. The pellet contains highly purified nucleoli.
Check under a phase-contrast microscope to ensure
that this preparation contains only highly purified
nucleoli without any other material (Fig. 5). Nucleoli
can be resuspended in 0.5ml of the S2 solution and
stored at -80°C.
|FIGURE 5 Step 9 of the procedure. Note the clear jboundary between S2 and S3 layers before and after
centrifugation. The pellet should be small but visible. (Insets) DIC images of the supernatant and pellet. The
pellet should contain purified nucleoli. Bars: 10µm (left inset) and 20µm (right inset).
1. Making 2.55 M Sucrose Stock
Here is a protocol for preparing a sucrose stock solution
(Cline and Ryel, 1971) suitable for the nucleolar
isolation protocol. The resulting solution is 2.55M
or 66% by weight. Its density is 1.3224g/cm3
at 20°C, and its refractive index is 1.4558. The stock solution
is stable indefinitely at 4°C. This procedure can be
carried out at room temperature. There is no need to
heat up the solution to help dissolve the sucrose.
Heating up an incompletely dissolved sucrose solution
can lead to charring of sucrose and affect the quality
of the sucrose solution.
- Weigh out 1710g sucrose (BDH). Keep it aside in a
- Put exactly 900ml water and a magnetic bar in a
5 litre beaker. Put the beaker on a stirrer and start
- Add one-third of the sucrose into the beaker. Make
sure the magnetic bar is rotating freely. Stir for 1 h.
- Add another one-third of the sucrose into the solution.
Again make sure the rotation of the stir bar is
not impaired. Stir for another 1 h.
- Add the remaining sucrose. Stir for another 1h or
until all the sucrose has gone into solution. The final
volume should be exactly 2 litres.
We use a Misonix 2020 sonicator fitted with a
microtip at power setting 5. To ensure reproducible
soncation, the following points should be followed.
3. Analysis of Isolated Nucleoli
- It is necessary to tune the sonicator every time
after you change the probe. Follow the manufacturer's
manual for the tuning procedure.
- Sonication produces intense and localized heat
in your solution. If you are concerned about the
heating, the correct way to reduce heating is to shorten
the sonication time and to increase the intermission
between bursts. Keeping the tube on ice or performing
the sonication in the cold room is helpful, but is not
the most effective way of heat control.
- If the probe is too close to the liquid surface, it
produces a foam and reduces the efficiency of sonication.
Make sure the probe is well submerged in the
solution, about 5 mm above the bottom of the tube. Do
not, however, touch the bottom or the wall of the tube
with the probe.
- A sonicator probe that has been used repeatedly
develops pits on its end. The sonication efficiency
gradually decreases as time goes on. Therefore, the
sonication time recommended here can only be used
as a guideline. Always monitor the outcome of sonication
using a phase-contrast microscope. You may need to adjust the sonication time to maintain the efficiency,
especially if the probe is getting old. Change the
probe when the efficiency is noticeably down.
4. Adapting Nucleolar Isolation Protocol to Use
with Other Cell Types
- To immunolabel purified nucleoli, spot about 5 µl
of the nucleolar suspension onto a polylysine-coated
slide (BDH Cat. No: 406/0178/00) and air dry the spot.
Rehydrate the slide in PBS for 5min before carrying
out a standard immunostaining procedure.
- To separate nucleolar proteins on a gel, resuspend
directly either in Laemmli SDS sample buffer
or in your preferred buffer. The high concentration
of nucleic acid in isolated nucleoli makes the lysed
sample very viscous. The sample can be clarified by
passing through a QIAshredder spin column (Qiagen
Cat. No: 79654). Nucleoli can also be extracted with
RIPA buffer (150 mM NaCl, 1% NP40, 0.5% deoxycholate,
0.1% SDS, 50mM Tris, pH 8.0, Complete protease
inhibitor cocktail). Immunoprecipitations can be
performed from nucleolar lysates prepared in RIPA
The aforementioned protocol can be adapted
readily to other cell types. Apart from HeLa cells, we
have used this protocol, with minor modifications, to
isolated nucleoli from MCF-7 (human breast epithelium),
WI-38 (human fibroblast), IMR-32 (human neuroblastoma),
HL60 (human promyelocytic leukemia),
and plant Arabidopsis thalina
cells. When adapting the
protocol to a different cell type, make sure that you
control each step by carefully checking the products after each step under a phase-contrast microscope. For
example, different cell types may require a different
homogenization (step 4) and/or sonication strength
(step 7). The concentration of MgCl2
crucial to the purity of the isolated nucleoli. If the isolated
nucleoli are not pure enough, try lowering the
concentration of MgCl2
in the S2 and S3 solutions. If
the yield is poor or if nucleoli look fragmented, use
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