In vitro Assays for Mitotic Spindle
Assembly and Function
During .the past two decades, cell biology has
entered a phase in which technology is so powerful
that fundamental questions concerning the morphogenesis
and function of cellular organelles can be
addressed. One essential and beautiful structure is
the mitotic spindle. The work of Lohka and Maller
(1985), followed by that of a few other laboratories
(Murray and Kirschner, 1989; Sawin and Mitchison,
1991; Shamu and Murray, 1992), opened up a novel
approach to studying such a complex and dynamic
structure. The idea is not to purify individual spindle
components and put them back together hoping that
a spindle will assemble, it is rather to open up the cell
and prepare g cytoplasmic extract as crude and concentrated
as possible to keep the conditions close to the in vivo
situation. At first sight, this approach seems
uninformative: one merely mimics in vitro
happens in vivo
. However, several methods have been
developed to manipulate the system, such as the addition
of reagents and depletion of proteins. Also the
microscopy techniques have evolved and now allow
the study of different aspects of spindle formation and
function (Desai et al.
, 1998; Kalab et al.
, 2002). Thus, this
system can be used both to analyze the mechanism of
spindle assembly and function and to evaluate the
role of individual molecules in the process. More
methods are still being developed or improved that
will increase the usage and the utility of these extracts
II. MATERIALS AND
A. Preparation of Xenopus laevis Egg Extracts
Incubator at 16°C, clinical centrifuge, DuPont
Sorvall RC-5 centrifuge, HB-4 or HB-6 rotor with
rubber adaptors (Sorvall Cat. No. 00363), Beckman
ultraclear SW50 tubes (Cat. No. 344057), Sarstedt
13-ml adaptor tubes (Cat. No. 55.518), 1-ml syringes,
18- and 27-gauge needles, glass pasteur pipettes.
Mature female frogs are from African Reptile Park.
Pregnant mare serum gonadotropin (Intergonan) is
from Intervet. Human chorionic gonadotropin (HCG,
Cat. No. CG-10), cytochalasin D (Cat. No. C-8273),
EGTA (Cat. No. E-4378), 4.9 M
(Cat. No. 104.20),
and ATP (Cat. No. A-2383) are from Sigma. CaCl2
No. 2383), KCl (Cat. No. 1.04936.1000), NaCl (Cat. No.
1.06404.1000), and L
-cysteine (Cat. No. 1.02838.1000)
are from Merck. Leupeptin (Cat. No. E18) and pepstatin
(Cat. No. E19) are from Chemicon. Aprotinin
(Cat. No. 981532) and creatine phosphate (Cat. No.
0621714) are from Roche. HEPES is from Biomol.
Sucrose is from USB (Cat. No. 21938).
B. Preparation of DNA Beads
Biotin-21-dUTP (Cat. No. 5201-1) is from Clontech.
Biotin-14-dATP (Cat. No. 19524-016) is from Invitrogen.
Thio-dCTP (Cat. No. 27-7360-02), thio-dGTP (27-
7370-04), and G-50 gel-filtration (NICK) columns (17-0855-01) are from Amersham Pharmacia Biotech.
Restriction enzymes and Klenow (Cat. No. M02125)
are from New England Biolabs. The magnetic particle
concentrator (MPC) and Kilobase BINDER kit containing
Dynabeads M-280 Streptavidin, washing, and
binding solutions can be obtained from Dynal. Trizma
base (Cat. No. T-1503) is purchased from Sigma, and
EDTA (Cat. No. 1.08418.1000) is from Merck.
C. Spindle and Aster Assays
Freshly prepared CSF extract, sperm nuclei
(3000/µl)(Murray, 1991), rhodamine-labelled tubulin
(2-3mg/ml) (Hyman et al.
, 1991), 76 × 26-mm microscope
slides, 22 × 22-mm coverslips, fluorescence
microscope. PIPES (Cat. No. P-6757), Triton X-100 (Cat.
No. T-8787), glutaraldehyde (Cat. No. G-5882), NaHB4
(Cat. No. S-9125), GTP (Cat. No. G-8877), and Hoechst
dye (bisbenzimide, Cat. No. H33342) are from Sigma.
Glycerol (Cat. No. 1.04093.2500) and formaldehyde
(Cat. No. 1.04003) are from Merck. Protein A dynabeads
(Cat. No. 100.02) are from Dynal. Dimethyl
sulfoxide (DMSO, Cat. No. 27,685-5) is from Sigma-
Aldrich, and Paclitaxel (taxol equivalent) (Cat. No.
P-3456) is from Molecular Probes. For sedimentation
and immunofluorescence experiments, corex tubes (15
ml) are equipped with plastic adaptors (home-made,
see Evans et al.
, 1985) to support 12-mm round coverslips.
The tubes are centrifuged in an HB-4 rotor containing
rubber adaptors, pGEX expression vectors are
from Amersham Pharmacia Biotech.
A. Preparation of X. laevis CSF Egg Extracts
Good protocols detailing the preparation of
Xenopus egg extracts and sperm nuclei have already
been published (Murray, 1991). We describe here only
the preparation of metaphase cytostatic factor-arrested
"CSF" extracts as optimized in our laboratory for
spindle assembly reactions.
- Pregnant mare serum gonadotropin (PMSG): Dissolve
in sterile water to a final concentration of 200
units/ml. Store at 4°C.
- Human chorionic gonadotropin (HCG): Dissolve in
sterile water to a final concentration of 2000 units/
ml. Store at 4°C.
- MMR: 100mM NaCl, 2mM KCl, 1 mM MgCl2,
2mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8.
Prepare a 20X MMR stock solution. Adjust pH
with NaOH, autoclave, and store at room temperature.
Before use make 1X MMR from the 20X
stock solution and adjust pH again if necessary.
- Dejellying solution: 2% L-cysteine, pH 7.8. Prepare
in water and adjust pH with NaOH. Make fresh
just before use.
- LP: dissolve leupeptin and pepstatin in DMSO to
a final concentration of 10mg/ml. Store in 50-µl
aliquots at -20°C.
- Aprotinin: Dissolve in sterile water to a final
concentration of 10mg/ml. Store in 50-µl aliquots
- Cytochalasin D: Dissolve in DMSO to a final
concentration of 10mg/ml. Store in 50-µl aliquots
- 20X XB salts: 2M KCl, 20mM MgCl2, 2mM CaCl2.
Filter sterilize and store at 4°C.
- XB: 1X XB salts containing 50mM sucrose and
10mM HEPES. Adjust pH to 7.7 with KOH.
Prepare fresh before use.
- CSF-XB: Prepare from XB buffer by adjusting
MgCl2 to 2mM and adding 5mM EGTA. To
prepare CSF-XB with protease inhibitors (CSF-XB
with PI), add LP stock solutions and aprotinin to a
final concentration of 10µg/ml.
- Energy Mix: 20X stock contains 150mM creatine
phosphate, 20mM ATP, 2mM EGTA, 20mM MgCl2. Store in 100-µl aliquots at -20°C.
Comments on Sperm Nucleus Preparation
- Inject four to six frogs subcutaneously with
0.5 ml (1000 units) PMSG each using 1-ml syringes and
a 27-gauge needle at least 4 days before planning to
make an extract. They should be used within 2 weeks
after the priming injection. The number of frogs
required depends on the quantity and quality of eggs.
Five milliliters of eggs (one SW50 tube) gives approximately
1ml of extract.
- Sixteen to 18h before use, inject frogs subcutaneously
with 0.25-0.5 ml (500-1000 units) HCG. Place
the frogs in individual boxes containing 500ml MMR
- Prepare all solutions before starting collecting the
eggs: 2 liters MMR, 500ml XB, 400ml CSF-XB, 100ml
CSF-XB with PI, and 500 ml of dejellying solution. Rinse
all glassware with distilled water (eggs stick to plastic
dishes). Cut off the end of a glass pasteur pipette and
fire polish it to make a wide-mouth pipette.
- Collect laid eggs in MMR. Frogs can also be
squeezed, which often gives the highest quality eggs.
Keep eggs from different frogs in separate batches.
Discard batches of eggs containing more than 5% of
lysed, ugly, or stringy eggs.
- Wash a few times with MMR to take away skin
and other detritus and remove bad eggs. Pour off
MMR and add 250ml dejellying solution. When laid,
eggs are enveloped in a transparent jelly coat and do
not pack closely together. Swirl the beaker frequently
and change the dejellying solution. After removal of
the jelly coat, eggs pack together. This takes about
5 min. Eggs left for too long in cysteine will lyse.
- Pour off the dejellying solution and add 500ml
MMR. Repeat the rinse one more time. After removal
of the jelly coat, the eggs become fragile. They lyse
easily and can activate if in contact with air. They must
always remain immersed in buffer. Remove all badlooking
eggs: white and puffy, flattened, or activated
ones (darker pole retracted), and those with mottled
- Wash three times with 100-200ml XB.
- Remove as much buffer as possible, keeping all
eggs immersed. Wash three times with 100-200ml
CSF-XB and finally keep eggs in CSF-XB with PI.
- Transfer eggs using the cut glass Pasteur pipette
to SW50 tubes containing 1ml CSF-XB with PI plus
3µl cytochalasin D (30µg/ml). Always immerse the
pipette tip in solution before expelling eggs to prevent
contact with air. Transfer the SW50 tubes to 13-ml
Sarstedt adaptor tubes, which contain 0.5 ml of water
to prevent the tubes from collapsing.
- Centrifuge in a clinical centrifuge at 16°C at
600g for 1 minute and then immediately increase the
speed to 1200g for an additional 30 s. After centrifugation,
remove all excess buffer from the top of the
packed eggs. Removal of buffer is critical to obtain a
- Place the tubes in an HB-4 or HB-6 rotor containing
rubber adaptors. Centrifuge at 10,500rpm for
15 min at 16°C to crush the eggs.
- Place the tubes on ice. A yellow lipid layer is at
the top of the tube. Underneath is the cytoplasmic
layer, then heavy membranes, and yolk particles at the
bottom of the tube. Wipe the sides of the tubes with a
tissue before piercing with a 18-gauge needle at the
bottom of the cytoplasmic layer. Slowly and carefully
aspirate the cytoplasm using a 1-ml syringe with the
needle opening facing upward. Remove needle from
syringe and carefully expel the cytoplasm into a 1.5-ml
tube. Place on ice.
- Add LP and aprotinin to 10µg/ml (1:1000 dilution
of stocks) and cytochalasin D to 20µg/ml (1:500
dilution of stock). Add energy mix (1:20 dilution of
stock) and mix gently.
Based on Gurdon (1976) and modified by Murray
(1991), our only modification is to mash fragments of testes between two frosted (rough) slides before filtering
through a cheesecloth mesh.
B. Preparation of DNA Beads
For preparation of DNA beads, linearized couple
plasmid DNA and fill in with nucleotides so that one
end contains biotinylated bases and the other thionucleotides
to inhibit exonuclease activity and the formation
of large aggregates of beads. Couple the DNA
to 2.8 µm magnetic beads. Incubation in the extract will
allow the association of chromatin proteins and the
consequent formation of chromatin (Heald et al.
- TE: 10mM Tris, 1 mM EDTA, pH 8. Adjust pH with
HCl, autoclave, and store at room temperature.
- Washing and binding solutions: Included with
Kilobase BINDER kit.
- Bead buffer: 2M NaCl, 10mM Tris, 1 mM EDTA, pH
7.6. Adjust pH with HCl and store at room
- Hoechst dye solution: Dissolve bisbenzimide in water
to a final concentration of 10mg/ml. Store in the
dark at 4°C.
- Prepare plasmid DNA by Qiagen column purification.
While the sequence of the DNA is not important,
the plasmid should be more than 5kb to
effectively induce chromatin assembly. Cut 50 µg of the
DNA with two restriction enzymes that have unique
sites in the polylinker to produce one short and one
long DNA fragment. One end of the long fragment
should terminate in an overhang containing Gs and Cs
and the other should contain only As and Ts (e.g., Not1, BamH1). See Fig. 1.
- Ethanol precipitate the DNA and resuspend in
25 µl TE. Quantify recovery by OD260 measurement.
- Prepare fill-in reaction in 70µl containing 1X
Klenow buffer, 30 µg DNA, 50 µM nucleotides (biotindATP,
biotin-dUTP, thio-dCTP, and thio-dGTP) and
20 units Klenow. Incubate for 2h at 37°C.
- Remove unincorporated nucleotides, following
instructions supplied with the Sephadex G-50 gel filtration
column (NICK column). The DNA is eluted
in a large volume (400µl), but the recovery is better
than with spin columns. Quantify recovery by OD260 measurement.
- Prepare coupling mix by combining 400µl
biotinylated DNA and 400µl binding solution. Set
aside 25 µl of the coupling mix for later evaluation of
- Prepare 3µl of streptavidin-conjugated dynabeads
for each microgram of DNA recovered, so
120µl for 30µg. Retrieve beads using the MPC
(magnet) and wash once with 5 volumes of binding
solution (600µl for 120-µl beads). Retrieve the beads
and resuspend them in coupling mix containing DNA.
- Incubate bead/coupling mixture for several
hours (or overnight) on a rotating wheel at 4°C.
- Retrieve the beads and save the supernatant.
Compare the OD260 of the supernatant to the sample
taken before coupling to determine the amount of
DNA immobilized. Typically two-thirds of the DNA is
coupled. Stain 1 µl of beads with 5µg/ml Hoechst dye
and observe them by fluorescence microcopy. The
beads should appear as very bright dots with no dark
patches. If the amount of DNA coupled in the first
round does not seem sufficient, incubate the beads a
second time with biotinylated DNA.
- Wash beads twice with washing solution and
then twice with bead buffer. After the last wash, resuspend
the beads in bead buffer so that the final concentration
of immobilized DNA is 1µg/5µl of beads.
Store at 4°C.
|FIGURE 1 Steps in preparation of DNA beads.
C. Spindle and Aster Assembly in vitro
Chromatin triggers spindle assembly in CSF
Xenopus egg extract. Experimentally there are two
ways to induce formation of the spindle: by the addition
of sperm nuclei (Desai et al.
, 1999) or DNA beads
to the extract (Heald et al.
, 1996). To decide on which
method to use it is important to understand the characteristics
of the two systems. Each sperm nucleus is
tightly associated to a centriole. In a very simple
process, half spindles can assemble around sperm
nuclei just by incubation of the sperm nuclei in extract.
Over time these half spindles fuse to form bipolar spindles.
If the extract is cycled through interphase the
DNA replicates and each centrosome duplicates. The
addition of fresh CSF extract sends the extract back
into mitosis, chromosomes with their paired kinetochores
condense, the duplicated centrosomes move
apart, and a bipolar spindle assembles. Anaphase can
be induced at this point by the addition of calcium to
DNA beads are made from any kind of plasmid
DNA that is long enough to induce chromatin assembly.
They are unlikely to assemble kinetochores, as centromere
sequences are not present. Spindle assembly
around DNA beads is achieved by first assembling
chromatin on the beads in an extract that is cycled
through interphase. The beads are then retrieved and
resuspended in fresh CSF extract. Using this method,
spindles assemble in the absence of kinetochores and
centrosomes. It is then ideal to study certain processes
of spindle assembly that are independent of these
A simplified method to study microtubule dynamics
and focusing of microtubule minus ends is the assembly
of asters in CSF extract. Asters can be assembled by
the addition of human centrosomes purified from KE37
lymphoid cells (Bornens et al.
, 1987), DMSO, or taxol to
the extract (Wittmann et al.
Steps for Spindle Assembly around Sperm Nuclei
(see Fig. 2)
- 10X calcium solution: 4 mM CaCl2, 100 mM KCl,
1 mM MgCl2. Store in aliquots at -20°C.
- 4.9M MgCl2 stock solution (Sigma)
- 10X MMR: See Section III,A.
- Hoechst dye solution: See Section III,B.
- Spindle fix: 48% glycerol, 11.1% formaldehyde,
5µg/ml Hoechst dye in 1X MMR. Always prepare
fresh on day of use.
- BRB80: 80mM PIPES, 1 mM MgCl2, 1 mM EGTA,
pH 6.8. Prepare a 5X BRB80 solution. Dissolve components
in sterile water and, while stirring, add KOH
pellets until the PIPES dissolves. Adjust to pH 6.8 with
KOH solution. Sterilize by filtration and store at 4°C.
- Dilution buffer: 1X BRB80 containing 30% glycerol
and 1% Triton X-100. Store at room temperature. Better
preservation of the spindles can be achieved by adding
0.25% glutaraldehyde to the dilution buffer. Use a full
vial of glutaraldehyde and store in aliquots at -20°C.
- Aster dilution buffer: BRB80 containing 10% glycerol,
0.25% glutaraldehyde, 1 mM GTP, 0.1% Triton X-
100. Use a fresh vial of glutaraldehyde to prepare
buffer and store in aliquots at -20°C.
- Cushion: 40% glycerol, BRB80. Store at 4°C.
- Aster cushion: 25% glycerol, BRB80. Store at 4°C.
- PBS: 137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.4 KH2PO4 mM, pH 7.4. Sterilize by autoclaving
and store at room temperature.
- Immunofluorescence buffer (IF buffer): PBS containing
2% BSA and 0.1% Triton X-100. Add azide and
store at 4°C.
- Mowiol: 10% Mowiol 4-88, 25% glycerol, and
0.1M Tris, pH 8.5. To prepare, mix 6g Mowiol with 6
g glycerol. Add 6 ml sterile water and stir for several
hours at room temperature. Add 12ml 0.2M Tris, pH
8.5, and place the mixture in a heating plate at 50°C for
10min with continues stirring. After the Mowiol dissolves,
centrifuge at 5000g for 15 min. Store in aliquots
- Before using an extract to assemble spindles, its
quality should be tested by setting up a "half spindle"
reaction. Combine 20 µl CSF extract, 0.2 µl rhodaminelabelled
tubulin and 0.8µl sperm nuclei (about 75
nuclei/µl extract) in a 1.5-ml tube and mix. The extract
can be mixed by gently moving the pipette in circles or by pipetting up and down, always avoiding making
bubbles. Incubate the reaction mixture at 20°C and
take a squash at 30-40min. To make a squash, using a
cut-off tip, transfer 1 µl of reaction mixture to a microscope
slide, carefully place 5 µl of spindle fix solution
on top, and gently place a 18 × 18-mm coverslip on top.
Analysis of the squash by fluorescence microscopy
should reveal half spindles with condensed chromosomes
and occasionally also spindles. There should be
no free microtubule nucleation.
- If the extract is good, proceed with the spindle
assembly protocol. For each reaction add on ice 0.2µl rhodamine-labelled tubulin and 0.8 µl sperm nuclei to
20 µl CSF extract in a 1.5-ml tube and mix. Incubate for
10 min at 20°C and then release extract into interphase
by the addition of 2 g110X calcium solution. Mix gently.
- Incubate for 80min at 20°C. Check that the
extract is in interphase by taking a squash as described
in step 1. If the sample is to be saved, seal the coverslip
to the slide with nail polish. At this stage, nuclei
should appear large, round, and uniform, and microtubules
should be long and abundant.
- At 90min postcalcium addition, add 20µl of
fresh CSF extract containing 0.2 µl rhodamine-labelled
tubulin to the reaction.
- Incubate further at 20°C. Take squashes (step 1)
at different time points to assess the stage of spindle
formation. During the incubation, prepare 15 ml Corex
tubes with plastic adapters, a round 12-mm coverslip,
and 5 ml of cushion.
- Forty-five to 60 min after mitosis reentry (step 4),
quickly add 1ml dilution buffer and mix by gently
inverting the tube a couple of times. Carefully layer the
mixture over the cushion using a cut pipette tip. Centrifuge
at 16°C for 12min at 12,000rpm in an HB-4 or
HB-6 rotor. Aspirate supernatant and cushion before
removing the coverslip. Fix coverslips in -20°C methanol for 5 minutes. Rehydrate samples by placing
the coverslips in PBS. If dilution buffer contains glutaraldehyde,
incubate the coverslips twice for 10min
in 0.1% NaHB4 in PBS and wash with PBS.
- To perform immunofluorescence, place the coverslip
face up on a surface covered with parafilm. Incubate
the coverslips with primary antibodies diluted in
IF buffer for 20-30 min. Wash the coverslips two times
for 5 min with PBS and incubate with IF buffer containing
the secondary antibody and 5µg/ml Hoechst
for 20-30 min. Wash three times for 5 min with PBS and
carefully place the coverslips upside down on a 3- to
4-µl drop of Mowiol on a microscope slide, avoiding
air bubbles. Prior to observation, allow the Mowiol to
set for 15min at 37°C or at room temperature for
- To visualize the early steps of anaphase, add 4 µl 10X calcium solution to the metaphase spindles before
step 6. Take samples before calcium addition and every
5 min after calcium addition for 20-30min and process
them as described earlier. It is not always possible to
visualize separation of the sister chromatids and only
high-quality extracts can be used successfully to study
|FIGURE 2 Preparation of CSF Xenopus egg extract and structures that assemble after addition of different
components. (1) Addition of sperm nuclei to the CSF extract leads to the formation of half spindles that
can fuse to form bipolar spindles. (2) Addition of calcium together with sperm nuclei sends the system into
interphase, allowing DNA replication and centrosome duplication to occur. Addition of fresh CSF extract
sends the system back to mitosis and bipolar spindles form. Calcium addition at this point induces chromosome
segregation and entry into interphase. (3) Bead spindle: addition of DNA-coated beads and calcium to
an extract leads to the formation of chromatin and nuclei around beads. Addition of CSF extract followed by
incubation in fresh CSF extract allows the formation of bipolar spindle. (4) Asters: Addition of purified centrosomes
or DMSO or taxol leads to the formation of microtubule asters with microtubule minus ends focused
at the centrosome or at the center of the aster.
Steps for Spindle Assembly around Chromatin Beads
(see Fig. 2)
Steps for Aster Formation (see Fig. 2)
- Transfer 3 µl of DNA beads (about 0.5 µg DNA)
to a 0.5-ml Eppendorf tube and place on a magnet.
Remove supernatant, and wash beads by resuspending
them in 20 µl of extract.
- Retrieve beads and resuspend in 100µl CSF
extract. Transfer to a 1.5-ml Eppendorf tube and incubate
- After 10min, release the CSF extract into interphase
by adding 10µl of calcium solution. Incubate for
2h at 20°C.
- Return the extract containing the beads to mitosis
by adding 50µl of fresh CSF extract. Incubate for 30
more min at 20°C. The procedure can be continued or
stopped at this point by freezing the beads in the
extract using liquid ethane and storing them in liquid
- Incubate the bead mixture on ice for several
minutes. Retrieve the beads on ice over 10-15 minutes.
Due to the viscosity of the extract, bead retrieval is
slow. Pipette the mixture every several minutes,
keeping the tube on the magnet.
- Remove the supernatant and resuspend the
beads in 150µl of fresh CSF extract containing 1.5µl
- Incubate at 20°C. Monitor the spindle assembly
by taking 1-µl samples and squashing with fixative as
described earlier. Spindle assembly requires between
30 and 90 min, depending on the extract. For immunofluorescence
studies, spin the bead spindles through a
cushion onto a coverslip and perform immunofluorescence
as described previously for spindle assembled
around sperm nuclei.
D. Functional Studies of Proteins Involved
in Spindle Assembly
- For each reaction, add on ice 0.2µl rhodaminelabelled
tubulin to 20µl CSF extract in a 1.5-ml tube.
To assemble asters, add either purified human centrosomes
(Bornens et al., 1987) or 5% DMSO or 1 µM taxol.
- Incubate the reaction for 30-60min in a 20°C. water bath. During this incubation, prepare as before
15-ml Corex tubes with plastic adapters, round 12-mm
coverslip, and 5 ml aster cushion.
- At the end of the incubation time, dilute the
reactions with aster dilution buffer and carefully layer
them on top of the cushion using a pipette with a cut
tip and subsequently centrifuge onto the coverslips in
a HB4 or HB6 rotor at 12,000rpm for 12min at 16°C.
- As before, remove the cushion with a vacuum
pump and postfix the coverslips in -20°C methanol for
5min. To quench the glutaraldehyde, incubate twice
for 10min in 0.1% NaHB4 in PBS and process for
The Xenopus egg extract system is a powerful tool
to assess the involvement of individual proteins in the
process of spindle assembly. Different methods have
been developed to address specific questions.
Localization studies can be performed by classical
immunofluorescence methods or by direct visualization
of GFP-tagged proteins added to the reaction
mixture or by a combination of both by adding GSTtagged
proteins followed by immunofluorescence with
Functional studies can be performed by adding
dominant-negative constructs to the reaction mixture
or by depleting the protein or antibodies under study
from the extracts before use (Antonio et al.
Boleti et al.
, 1996). Depletion experiments should be
complemented by "add back" or rescue experiments
in which a recombinant protein is added to the
depleted extracts (Fig. 3). In all tree cases, a careful
quantification of the structures found in control or
treated samples has to be performed.
|FIGURE 3 Spindles assembled in CSF Xenopus egg extract following
the cycled spindle pathway. The extract was mock depleted,
Xkid depleted, or Xkid depleted supplemented with recombinant
Xkid protein (add back). When Xkid was depleted, mitotic chromosomes
failed to align at the metaphase plate. Alignment of chromosomes
was rescued by the addition of recombinant protein to the
extract. Scale bar: 10 µm.
Steps for Immunodepletion
- CSF-XB with PI: See Section III,A.
- PBS-TX: PBS containing 0.1% Triton X-100. Store at 4°C.
Steps for Specific Antibodies or Dominant-Negative Protein Addition
- Usually around 10µg of specific antibody is
needed to deplete 150 µl of extract. The amount of antibody
has to be adjusted for each case depending on
the antibody itself and on the abundance of the protein
in the extract. Prepare a control mock-depleted extract
using the same amount of unspecific purified IgG.
- Transfer 40 µl of Dynal beads coupled to protein
A to a 1.5-ml tube and retrieve them using a magnet.
Wash them twice by resuspending the beads in 500µl
PBS-TX and retrieving them with the magnet.
- Incubate the beads with 10 µg of antibody diluted
in 200 µl of PBS-TX for 1h on a rotating wheel at 4°C.
- Wash twice as just described with PBS-TX and
once with CSF-XB containing PI. Remove as much wash
buffer as possible and carefully resuspend the beads in
150µl of extract by pipetting up and down. Keep the
extract (containing the beads) on ice for 90min with
occasional mixing by pipetting up and down.
- Retrieve the beads by placing the tube on the
magnet for 5 min on ice. Collect the extract into a new
tube and keep it on ice. To remove the beads more efficiently,
repeat this procedure once more. Check the
efficiency of depletion by Western blot analysis of
mock and protein-depleted extracts. To visualize the
immunoprecipitated protein, wash the beads once
with CSF-XB containing PI and three times with PBSTX.
Resuspend the beads in Laemmli sample buffer
and incubate at room temperature for 5 min. Retrieve
the beads on a magnet and boil the supernatant before
loading on SDS-PAGE.
- An important control to include in depletion
experiments is the add back of the purified recombinant
protein to the depleted extract in a range of concentrations
close to that of the endogenous protein. In
case the protein is degraded during interphase, the recombinant protein should be added only at mitosis
- The depleted, the control mock depleted, and
add-back extracts can now be used in parallel to
assemble asters and spindles following the methods
described in Section III,C.
- Compare the spindles or asters formed in each
reaction either by taking samples and squashing them
in a slide with fixative solution or by centrifuging them
onto coverslips and performing immunofluorescence.
- In the case of addition of specific antibodies,
prepare a concentrated solution (at least 2mg/ml) of
an affinity-purified polyclonal or monoclonal antibody
in PBS or CSF-XB. It is a good idea to try different concentrations
of antibody (ranging from 200 to 400µg/
ml). For dominant-negative addition, prepare constructs
expressing different fragments of the protein
under study fused to GFP or GST. Purify the fusion
proteins and dialyze them extensively against CSF-XB.
- Add the antibodies or dominant-negative
protein to the extract in a volume corresponding to
one-tenth of the total volume and follow the protocol
for spindle assembly or aster formation as described in
Section III,C. The antibodies or dominant negative can
be added at the beginning of the spindle assembly procedure
or at mitosis reentry.
- When adding antibodies, two control samples
should be run in parallel: one containing a similar
amount of a control antibody and another containing
the same volume of buffer. In the case of dominantnegative
addition, include also the same volume of
buffer as control and a sample containing GST or GFP
- Samples taken at different time points are fixed
and analyzed as before. To examine the localization of
the antibody, process the samples by immunofluorescence
using a fluorochrome-conjugated secondary
antibody. Localization of the dominant negative can be
observed directly if the tag is GFP or by using an anti-
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every time, which can be frustrating. Therefore, experiments
must be repeated several times to ensure a
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