We recommend that you first download and read this page as a PDF
file. Using that as your guide, you can then follow the protocol
below and view a Quicktime movie demonstrating the key steps. We have
also included higher resolution stills which provide close-ups of certain
steps of the protocol.
|Special Feature: Complete nucleolar isolation
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|The movie stars Dr.
Yun Wah Lam, who optimized the original protocol.
Buffers and solutions
(All solutions are supplemented with Complete Protease inhibitor tablet
(Roche, Cat no: 1-873-580) at the final concentration of 1 tablet/50ml):
Buffer A: 10 mM Hepes, pH 7.9, 10mM KCl, 1.5mM MgCl2,
S1 solution: 0.25 M Sucrose, 10 mM MgCl2
S2 solution: 0.35 M Sucrose, 0.5 mM MgCl2
S3 solution: 0.88 M Sucrose, 0.5 mM MgCl2
See “Notes” below on making stock sucrose solution.
Note: Nucleoli were prepared from HeLa cells using a variation on a method
described by Muramatsu and co-workers in 1963 (Muramatsu M, Smetana,
K., and Busch, H.: Quantitative aspects of isolation of nucleoli of the
Walker carcinosarcoma and liver of the rat. Cancer Res. 1963; 25:693-697).
1. Seed HeLa cells (ATCC number: CCL-2) on to 10x14 cm Petri dishes and
culture at 37oC 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 100 U/ml Penicilin
and 100 µg/ml Streptomycin (1% v/v Penicilin/Streptomycin solution,
Invitrogen UK, Cat no: 15140-122) until >90% confluence (approx. 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 purity of the isolated nucleoli may suffer. Make sure you monitor
every step using a phase contrast microscope (see below). 1 hour before
nucleolar isolation, replace with fresh, pre-warmed medium.
2. Harvest cells by trpysinization. Rinse each dish 3X with pre-warmed
PBS, and on removal of the last rinse, add 2 ml of trypsin-EDTA solution
(Invitrogen UK, Cat no: 25300-054) per dish. Swirl the dishes to make
sure the trypsin-EDTA is evenly distributed, and return the dishes to
the incubator for about 5 min. Check under a phase contrast microscope
that all the cells are detached. Prolong incubation if needed. Add into
each dish 8 ml of pre-warmed medium, pipette up and down so that all the
cells are collected as a single-cell suspension. Pool all the harvested
cells into 2x 50ml 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. Since scraping may lead to impure nucleolar preparation
in some HeLa strains, it is not recommended as the method of first choice.
3. Wash 3 X with ice-cold PBS at 218 g (1000 rpm, Beckman GS-6 centrifuge,
GH-3.8 rotor) at 4oC.
4. After the final PBS wash, resuspend the cells in 5ml 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 20X objective. The cells should be swollen,
but not burst (Fig 1). Nucleoli of cultured mammalian cells disassemble
at 37oC 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: 10µm.
5. Transfer the cell suspension to a pre-cooled 7 ml Dounce
tissue homogenizer (Wheaton Scientific Product Cat no: 357542). Homogenize
10 times using a tight pestle (“A” specification: 0.0010"
- 0.0030" clearance), while keeping the homogenizer on ice. The number
of strokes needed depends on the cell type used (see “Notes”).
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 (Figure 2).
6. Centrifuge the homogenized cells at 218g (1000 rpm, Beckman
GS-6 centrifuge, GH-3.8 rotor) for 5 min at 4°C. The pellet contains
enriched, but not highly pure, nuclei.
7. Resuspend the pellet with 3 ml S1 solution (Figure 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 3 ml of S2 solution. Take care to keep the two
layers cleanly separated. Centrifuge at 1430g (2500 rpm, Beckman GS-6
centrifuge, GH-3.8 rotor) for 5 min at 4°C. This step results in a
cleaner nuclear pellet (Figure 3). Resuspend the pellet with 3 ml of 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
show the DIC images of the supernatant and pellet. Note the prominent
nucleoli inside the nuclei in the pellet. Bars: 10µm.
8. Sonicate the nuclear suspension for 6 x 10 second bursts
(with 10 second intervals between each burst) using a Misonix XL 2020
sonicator fitted with a microtip probe and set at power setting 5 (Figure
4 Left). Check the sonicated nuclei under a phase contrast microscope.
There should be virtually no intact cells and the nucleoli should be readily
observed as dense, refractile bodies (Figure 4 right). 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 10 seconds of sonication. Over-sonication
leads to destruction of nucleoli.
Figure 4. Left. Setup for sonication. Right.
DIC image of sonicated nuclei. Note the presence of prominent nucleoli.
9. Layer the sonicated sample over 3 ml of S3 solution and
centrifuge at 3000g (3500 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor)
for 10 min at 4°C (Figure 5). The pellet contains the nucleoli, whilst
the supernatant can be retained as the “nucleoplasmic fraction”
Figure 5: Step 9 of the procedure. Note the
clear boundary between S2 and S3 layers before and after centrifugation.
The pellet should be small but visible. Insets show DIC images of
the supernatant and pellet. The pellet should contain purified nucleoli.
Bars: Left inset: 10µm, right inset: 20µm.
10. Resuspend the nucleoli with 0.5 ml of S2 solution, followed
by centrifugation at 1430 (2500 rpm, Beckman GS-6 centrifuge, GH-3.8 rotor)
for 5 min at 4°C. The pellet contains highly purified nucleoli. Check
under a phase contrast microscope to ensure this preparation contains
only highly purified nucleoli without any other material (Fig 5). The
nucleoli can be resuspended in 0.5ml of S2 solution and stored at –80oC.
(1) Making 2.55M sucrose stock
Here is a protocol for preparing a sucrose stock solution (Cline and Ryel,l
1971) suitable for the nucleolar isolation protocol. The resulting solution
is 2.55M, or 66% by weight. Its density is 1.3224g/cm3 at 20oC, and refractive
index is1.4558. The stock solution is stable indefinitely at 4oC. This
procedure can be carried out at RT. There is no need to heat up the solution
to help dissolving the sucrose. Heating up an incompletely dissolved sucrose
solution can lead to charring of sucrose and affect the quality of the
1. Weigh out 1710 g sucrose (BDH). Keep it aside in a clean container.
2. Put exactly 900ml water and a magnetic bar in a 5 litre beaker. Put
the beaker on a stirrer and start stirring.
3. Add 1/3 of the sucrose into the beaker. Make sure the magnetic bar
is rotating freely. Stir for 1 hour.
4. Add another 1/3 of the sucrose into the solution. Again make sure the
rotation of the stir bar is not impaired. Stir for another 1 hour.
5. Add the remaining sucrose. Stir for another 1 hour, 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 these points should be followed:
- 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 5mm above the bottom of the tube. Do
not, however, touch the bottom or the wall of the tube with the probe.
- 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 reecommended here can only be used as 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
(3) Analysis of the isolated nucleoli
- To immunolabel the purified nucleoli, spot about 5 µl of the nucleolar
suspension on to a polylysine-coated slide (BDH Cat no: 406/0178/00),
and air dry the spot. Rehydrate the slide in PBS for 5 min before carrying
out a standard immunostaining procedure.
- To separate nucleolar proteins on a gel, either resuspend directly in
Laemmli SDS sample buffer or in your preferred buffer. The high concentration
of nucleic acid in the 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, 50 mM Tris pH 8.0,
COMPLETE protease inhibitor cocktail). Immunoprecipitations can be performed
from nucleolar lysates prepared in RIPA buffer.
(4) Adapting nucleolar isolation protocol to use with
other cell types
The above protocol can readily be adapted 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 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 also appears 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 the nucleoli look fragmented, use
(5) Preparing highly purified nuclei (i.e. stop before you get
We use a shortened version of the nucleolar purification protocol to prepare
highly purified nuclei. These can be solubilized in RIPA buffer and sonicated
to release > 75% of nuclear proteins. In short, trypsinize, pellet
and wash cells, and incubate in Buffer A. Dounce 10x and spin to pellet
nuclei (note, up to this step it's the same as the nucleolar protocol).
Pipette off the cytoplasm. Resuspend nuclear pellet in 3 ml of S1 and
layer over 3 ml of S3. Centrifuge at 3000g (3500 rpm, Beckman GS-6 centrifuge,
GH-3.8 rotor) for 15 min at 4°C. Remove supernatant and keep nuclear
pellet. One way to release nuclear proteins is to then resuspend this
pellet in 3 ml RIPA buffer and sonicate 6 x 10 sec bursts on ice, with
10 sec rest on ice in between. Centrifuge again at 3000g for 10 min to
pellet any residual solids, and pipette off the supernatant, which is
your nuclear lysate.