Isolation of Cajal Bodies
The Cajal body (CB) is a subnuclear organelle
involved in the biogenesis of snRNPs and snoRNPs. It
was first described by Ramon-y-Cajal as the "nucleolar-
accessory body" in silver nitrate-stained neuronal
cells (Cajal, 1903). Later, electron microscopy studies
on neuronal cells showed that this nuclear domain
sometimes resembled a ball of coiled threads 0.15 to
1.5 gm in diameter. The identification of CBs in the fluorescence
microscope was facilitated by the discovery
of p80 coilin, a human autoantigen that is enriched
in the CB (Andrade et al.
, 1991). Antibodies against
human p80 coilin label CB-like nuclear foci in a wide
spectrum of organisms, showing that the CB is a highly
conserved structure. While various lines of experiment
suggest a role of the CB in snRNA and snoRNA
maturation (reviewed by Gall, 2000), CBs also contain
proteins involved in other pathways, such as nucleolar
functions, tumourigenesis, and cell cycle regulation
(Sleeman and Lamond, 1999; Jacobs et al.
, 1999; Liu et al.
, 2000, Ma et al.
, 2000). Therefore, CBs may also
have other cellular functions that remain to be
This article describes an effective procedure for the
large-scale isolation of CBs from mammalian somatic
cell nuclei (Lam et al.
, 2002). The procedure involves
disrupting HeLa cell nuclei by sonication, treatment
with detergent, nuclease, and polyanion, and subsequent
density gradient fractionation. Density separation
is carried out using Percoll, a silica sol coated with
polyvinylpyrolidone, which generates a density gradient
on ultracentrifugation. It results in the enrichment
of particles containing known CB factors that are comparable
in size, morphology, and composition to CBs
detected in situ
. These particles, therefore, correspond
to isolated CBs. Judging by both immunofluorescence
and immunoblotting analyses, we estimate that this
protocol provides an enrichment factor of at least 750-
fold for isolated CBs, as compared to intact nuclei. The
protocol is reproducible and can be carried out on a
sufficiently large scale to generate sufficient material
for biochemical analysis.
II. BUFFERS AND SOLUTIONS
All solutions are supplemented with Complete protease
inhibitor tablet (Roche, Cat. No. 1-873-580) at a
final concentration of 1 tablet/50ml).
sucrose and 10 mM
sucrose and 0.5 mM
sucrose and 25 mM
Tris-HCl, pH 9.0
sucrose, 34.2% Percoll, 22.2mM
Tris-HCl, pH 7.4, and 1.11 mM
: 20% Percoll, 10mM
Tris-HCl, pH 7.4, 1%
Triton X-100, and 0.5mg/ml heparin
: 10 mM
Tris-HCl, pH7.4, 1% Triton ×100, and
0.5 mg/ml heparin
See Section IV on making sucrose stock solution.
All procedures should be performed either on ice or
- Prepare the starting material of 2.5 × 109 HeLa
nuclei. Suitable HeLa nuclei can be purchased from
CIL Biotech (Mons, Belgium; Cat. No. CC-01-30-25).
Add S1 solution to thawed nuclei to a final volume of
30ml. Mix thoroughly but avoid vigorous vortexing.
- Divide the diluted nuclei into 2 × 15 ml portions.
Overlay each portion onto 15 ml of the S2 solution in
a 50-ml Falcon tube. Make sure the interface of the two
layers is sharp (Fig. 1).
- Centrifuge at 1430g (2500rpm, Beckman GS-6
centrifuge, GH-3.8 rotor) for 5 min at 4°C.
- Carefully decant the supernatant. The pellets
should be solid and firm (Fig. 1). Resuspend each
pellet with the S2 solution so that the final volume is
30 ml. The pellets should resuspend easily. A pellet that
cannot be resuspended completely indicates the presence
of lysed nuclei and should be discarded.
- Divide resuspended nuclei into 10 × 3-ml portions,
each in a 15-ml Falcon tube. In our experience,
sonication is more effective with this sample size.
- Sonicate each aliquot using a tuned sonicator
(Misonix XL 2020) for 3 × 6s, with a 6-s intermission
between each burst. See Section IV on sonication.
- Examine the sonicated nuclei under a phasecontrast
microscope. The majority of nuclei should
have been lysed, while nucleoli should be clearly
visible. If the percentage of unlysed nuclei is too high
(over 5%), sonicate again for 2-3s and then reexamine
under the microscope. Stop at the point when most
nuclei are lysed. Over sonication disrupts CBs. The
conditions used to sonicate HeLa nuclei not only maintain
the structure of CBs, but also that of nucleoli (see Isolation of Nucleoli, this volume). PML bodies, and splicing
speckles. This indicates that, under suitable conditions,
many nuclear domains remain intact even after
the overall nuclear structure is destroyed.
|FIGURE 1 Steps 2-3 of the procedure. Note the clear boundary between S1 and S2 layers before
B. Removal of Nucleoli
- Pool the 10 aliquots back together. Measure the
volume. Add 0.42× volume of 2.55M sucrose to sonicated
nuclei so that the final sucrose concentration is
now 1M. Mix thoroughly but do not vortex. Divide the
mixture into two portions, approximately 20ml each,
in two 50-ml Falcon tubes (Fig. 2).
- Centrifuge at 3000g (3500rpm, Beckman GS-6
centrifuge, GH-3.8 rotor) for 10min at 4°C.
- A small pellet should be visible in each tube
(Fig. 2). This pellet contains nucleoli and unlysed
nuclei, while the supernatant is nucleoplasm containing
CBs. Using a pipette, collect the supernatant
carefully. Do not decant, as the pellets are not firm.
Stop when you are about 5 mm above the pellet. This
is the region where the distinction between the pellet
and the supernatent is blurred. This part of the supernatant
should not be included in subsequent steps.
- To collect nucleoli for other experiments, resuspend
the pellet with 3 ml S2. Sonicate the mixture for
3 × 10s. Proceed from step 7 of the nucleolar isolation
protocol (see article by Lam and Lamond).
|FIGURE 2 Steps 8-10 of the procedure. Note the small pellet after centrifugation. (Right) Microscope
images of the supernatant and pellet. Anticoilin antibodies (5P10, green) and pyronin Y (red) were used to
label CBs and nucleoli, respectively. Bar: 10µm.
C. Gradient One
- Measure the volume of the supernatant. Add
0.82× volume of SP1. For example, in a typical experiment,
mix 37 ml of the supernatant with 30.27 ml SP1.
Add 0.05x volume of 20% Triton X-100 (3.36ml in the
aforementioned example). Mix thoroughly but do not
- Divide the mixture into precooled SW41 ultracentrifuge
tubes (Fig. 3). The volume of the sample
should fit six tubes perfectly. Balance the tubes carefully.
Load the tubes into a precooled SW41 rotor.
Transfer the rotor to a precooled ultracentrifuge.
- Ultracentrifuge at 37,000rpm for 2h.
- A loose pellet can be seen resting on the Percoll
precipitate at the bottom of each tube (Fig. 3). This
pellet contains most of the Cajal bodies. Carefully
pipette away the turbid supernatant. Do not decant.
Stop when you are 5-6 mm above the pellet. Keep the
supernatant labeled as fraction 1S. Use a P1000 pipette
and resuspend the pellets in the remaining supernant.
Transfer the resuspended pellets to a 15-ml Falcon
tube. This is fraction 1P. Measure the final volume
of fraction 1P. In a typical experiment, the final volume
is about 5ml. In fraction 1P, the CBs present are
mostly entangled with large pieces of chromatin, as
revealed by DAPI staining. Nucleoli that have not
been removed in the first step are also a significant
|FIGURE 3 Steps 13-15 of the procedure. Note the loose pellet (1P) after centrifugation.
(right) shows that most CBs are in 1P. Bar: 10µm.
D. Gradient Two
- Add 600 units of DNasel (Sigma Cat. No.
D4527; 20,000 units/ml) to fraction 1P. Mix with a
rotating wheel for 1 h at room temperature.
- Add 0.05× volume of heparin (10mg/ml). The
mixture should become more transparent on mixing.
- Add 1× volume of SP2. Mix gently with a rotating
wheel for 1 min.
- Load the mixture in precooled tubes suitable for
SW55 rotor (Fig. 4). In a typical experiment, the
mixture fits two tubes perfectly. Ultracentrifuge at
45,000 rpm for 1 h.
- Three bands should be visible (Fig. 4). A broad
white band floats on the top, a thin white band at
the middle, and a pellet resting on the Percoll precipitate
at the bottom. Using a P1000 pipette, very
carefully unload the gradient from the top. Take
the top band and go on until you are 2-3 mm above
the middle band. This is fraction 2.1. Using a new
pipette tip, take the middle band (fraction 2.2). Finally,
resuspend the pellet using the remaining part of the
gradient. This is fraction 2.3. For each 5-ml gradient,
typical volumes of the three fractions are fraction
2.1, 2.5ml; fraction 2.2, 1 ml; and fraction 2.3, 1.5 ml.
Most of the CBs are contained in fraction 2.2 in the
middle of the gradient, where the resolution is the
|FIGURE 4 Steps 19 and 20 of the procedure. Note the thin band (2.2) in the middle of the gradient after centrifugation. Bar: 10µm.
E. Concentration and Final Enrichment of
- Add 10× volume of HT buffer into fraction 2.2.
Mix well using a rotating wheel.
- Either load the diluted fraction into 2 × SW41
tubes and ultracentrifuge at 12,000rpm for 15 min or
divide the mixture into 20 × 1.5-ml Eppendorf tubes
and centrifuge at 14,000rpm for 15min in an Eppendorf
centrifuge (Fig. 5).
- Remove the supernatant carefully. Resuspend
the pellet in 0.5 ml HT buffer. Pool all the resuspended
pellets into a single tube and centrifuge again as
described earlier. The final pellet should be visible, but
very small. Remove the supernatant carefully.
- Resuspend the pellet with 0.5ml S3 solution.
The pellet detaches from the bottom of the tube and is
resuspended on pipetting up and down.
- Spin down the resuspended pellet in an Eppendorf
centrifuge at 8000rpm for 5 min. A visible pellet
should be seen. Carefully take the supernatant and
transfer it to a new Eppendorf tube. Repeat spinning
the supernatant at 8000rpm for 5 min. Carefully take
the supernatant and transfer it to a new Eppendorf
- Combine the pellets. Label this fraction 3P.
- Dilute the supernatant with 10× volumes of
25mM Tris-HCl, pH 9.0. Divide the diluted supernatant
into 3 x 1.5-ml Eppendorf tubes. Centrifuge at
14,000rpm for 15 min. Remove the supernatants carefully,
leaving about 0.1 ml at the bottom of each tube.
Pipette the remaining supernatants up and down,
resuspending the small pellet. Mix the contents of the
three tubes together. Fill up the tube with 25mM Tris-HCl, pH 9.0, and centrifuge again at 14,000rpm
for 15 min. Remove the supernatant carefully, leaving
about 20µl in the tube. Pipette the remaining supernatants up and down to resuspend the small pellet.
Label this fraction 3S. This fraction contains enriched
|FIGURE 5 Steps 22-27 of the procedure. Most CBs are found in fraction 3S. (Inset) Arrows indicate the
presence of a small amount of coilin-negative material in 3S. Bars: 25 µm (large inset) and 10 µm (small insets).
1. Making 2.55M 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
66% by weight. Its density is 1.3224 g/cm3
at 20°C, and
the 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 1710 g sucrose (BDH). Keep it aside in a
- Put exactly 900ml water and a magnetic bar in a
5-1itre 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 1 h 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 the Enriched Cajal Body Fraction
- 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 the 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.
- To immunolabel the isolated CBs, spot about 5 µl of
fraction 3S onto a polylysine-coated slide (BDH Cat.
No. 406/0178/00) and air dry the spot. Rehydrate
the slide in PBS for 5 min before using the standard
- To separate and analyse the proteins by SDS-PAGE,
resuspend directly either in Laemmli SDS sample
buffer or in your preferred buffer.
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