Preparation of Tubulin from
In the previous edition to this series (2nd Ed., Vol.
2, pp. 205-212, 1998), we described a method to purify
tubulin from bovine brains. Since that time there have
been concerns in many countries over the risks of handling
bovine nervous tissue relating to bovine spongioform
encephalitis and its possible connection with
human varient Creuzfeldt-Jakob disease. As a result,
abattoirs have been reluctant or have even refused to
handle these tissues. Consequently we have changed
the species source from which we make the tubulin
and have amended the title accordingly. However the
protocols described are applicable in their entirety to
the bovine source. The bovine and porcine equivalents
are indicated in the protocol.
Recent research on microtubules has given much
insight into many fundamental problems in cell
biology, such as cell structure and polarity, vesicular
transport, cell division, and chromosomal segregation.
Furthermore, the ability to prepare polarity marked
microtubules (Howard and Hyman, (1993) has contributed
much to our understanding in the behavior of
Microtubules are polymers of the heterodimeric
protein tubulin. Because these heterodimers have the
same orientation in the microtubule lattice and the
dimers themselves are not symmetric, it follows that
the microtubule itself must have an inherent polarity.
Polymerization can take place from either end of the
growing microtubule, although at different rates,
having a slow growing (-) and more rapidly growing
(+) end. The tubulin heterodimer has binding sites for
two GTP molecules in its unpolymerized state, only
one of which (that GTP that is bound to the E site of
the tubulin β subunit) is hydrolyzable. While it is not
within the scope of this article to examine all the properties
of microtubules and tubulin, some of these are
of direct relevance to their isolation. The current model
first proposed by Mitchison and Kirschner (1984a) and
developed further since then (reviewed by Hyman
and Karsenti ) suggests that GTP-unpolymerized
tubulin is incorporated at the + growing end of
the microtubule and that after a delay, during which
time more GTP-bound tubulin is bound at the growing
end, the GTP on the β subunit is then hydrolysed to
GDP, possibly destabilizing the microtubule lattice.
Because GTP-bound tubulin dissociates from the
microtubules slowly and the dissociation rate of
exposed bound GDP tubulin is two or three orders of
magnitude higher, it follows that the presence of a socalled
GTP-tubulin "cap" will protect the polymerized
microtubule from depolymerization. Alternatively, the
GTP "cap" structure may be inherently more stable,
effectively preventing the GDP (destabilized) polymerized
tubulin from dissociating from the lattice.
Thus the "GTP cap" hypothesis states that if the microtubule
loses this GTP "cap" structure because of slow
growth (e.g., due to lack of free tubulin, cold temperature,
presence of Ca2+
), the GDP-tubulin dissociates
from the lattice. Microtubules growing more slowly
are more likely to have GDP-tubulin exposed and thus
dissociate rapidly, causing microtubule shrinkage.
The preparation of tubulin described here utilizes
these phenomena by a series of alternate depolymerizing
and polymerizing steps (see Fig. 1) to obtain tubulin and microtubule-associated proteins (MAPs),
followed by ion-exchange chromatography to separate
the tubulin from MAPs. This protocol is based on that
originally described by Shelanski et al.
(1973) and modified
by Weingarten et al.
(1974) and subsequently by
Mitchison and Kirschner (1984b). Tubulin can be modified
further for other uses (Hyman et al.
|FIGURE 1 Summary of tubulin preparation.
This article describes how to make pure tubulin
with emphasis on large-scale (gram quantities) purifications.
This, as will be made clear in later sections, is
quite an exercise in both organization and logistics.
However, the method can easily be scaled downwards
if large amounts of tubulin are not required.
Because the principle of tubulin preparation relies
on successive rounds of polymerisation and depolymerisation
steps based on temperature shifts, delays
and not having the right equipment at the right
temperature will reduce the yield drastically. Consequently,
after one centrifuge run is completed, it is
good practice to reset the temperature for the next spin
and switch on the vacuum, checking the centrifuge
regularly for faults. This also applies to the rotors, and
it is a good idea to have some large water baths set at
37°C and some ice troughs in which to either raise or
lower the rotor temperature accordingly. For the polymerization
of tubulin, a good supply of hot running
water is essential, as slow polymerization will reduce
the overall yield.
II. MATERIALS AND
The protocol, as stated earlier, is suitable for preparation
of large (2 to 3 gram) quantities of pure tubulin.
To obtain this quantity, it will be necessary to obtain
approximately 30 porcine (or 12 bovine) brains. It
follows from this that the volumes involved in processing
this number of brains, at least in the initial
stages of preparation, will be quite large and many
centrifuges will be necessary.
A. Centrifuges, Rotors, and Bottles
It is desirable, although not absolutely necessary, to
have a centrifuge capable of spinning, at low speed, up
to 10 litres of homogenate.
At least six Sorval RC5s, evolutions, or equivalent
centrifuge series with SuperLite SLA 1500 are needed
together with 36 GSA rotor bottles (250-ml capacity)
and their adaptors. The Beckman equivalent to this is
the Beckman Avanti using the JLA-16.250 rotor.
Four ultracentrifuges with three Beckman type 19
rotors and 24 corresponding bottles are needed along
with four Beckman type 45Ti rotors and 24 (70-ml
capacity) polycarbonate bottles.
As can be seen from this list, the rotors required are
quite large and heavy and it is therefore important to
check the vacuum and refrigeration efficiency of all the
centrifuges beforehand using these rotors. Regarding
the rotors themselves, these should be inspected
beforehand, and any seals and "O" rings should be
checked for signs of perishing, replacing where necessary.
It is also important that these "O" rings are
greased lightly with vacuum grease and that the screw
threads for the rotor lids are smeared lightly with Beckman "Spinkote" to ensure that the samples are
sealed and that the vacuum is maintained during the
run. Finally, the centrifuge rotors should be checked
for the condition of the overspeed decals to prevent
premature termination of the run. This is particularly
true of the Beckman type 19 rotors, which seem especially
sensitive on this point.
The centrifuge bottles too should be inspected for
cracks and warping (particularly true of the polycarbonate
Beckman type 45 bottles), and the lids and caps
should be checked for condition and integrity of the
B. Other Equipment
A tissue blender, such as that made by Waring,
along with a 4.5-1itre capacity homogenizing beaker
and a (motor-driven) continuous flow homogenizer,
such as the Yamato LH-22 model for resuspending the
tubulin pellets (or a very large Dounce hand homogenizer),
as well as a 1-Litre capacity phosphocellulose
(PC) column, are needed. Details of how to prepare
such a column are described in detail later.
All chemicals should be of analytical grade and can
be obtained from a variety of suppliers. Chemicals for
the following solutions are obtained from the following
MES (M-5057), PIPES (P-6757), EGTA (E-4378),
(104-20), GTP (G-8877), ATP (A-7699), and β-
mercaptoethanol (M-6250) are from Sigma-Aldrich.
EDTA (1.08418.1000), anhydrous glycerol (1.04093.
2500), NaCl (1.06404.1000), NaOH (1.06498.1000), HCl
(4873.1000), and K2
1000) are from Merck.
A. Preparation of Tubulin
The buffer systems of choice for the preparation of
tubulin are 2-[N
-MES for the blending buffer (BB) and
PIPES for the remainder. When making up K.PIPES
buffer solutions, it is first necessary to raise the pH of
the solution to about pH 6.0 using KOH in order to get
the PIPES to go into solution. This initial pH adjustment
must be done without the use of a pH meter
using pH indicator paper), as any undissolved PIPES
can damage the electrode.
Approximately 6 litres of BB is needed. Blending
buffer is 0.1M
MES, pH 6.5, 0.1 mM
EGTA, and 0.5 MgCl2
. For 1 litre, use 21.7 g MES, 4ml
EDTA stock solution, 0.1 ml 0.5M
solution, and 0.5 ml 1 M
About six litres of polymerization buffer (PB) is
needed for a large tubulin preparation; if preferred, it
can be made as a 5× stock solution and then diluted
with water at 4°C before use. Polymerization buffer is
PIPES, pH 6.8, 0.5 mM
, 2.0 mM
Approximately five litres of Column buffer
needed for such a preparation, which is made up as a
10× solution and diluted with water at 4°C prior to use.
Column buffer is 50mM
PIPES, pH 6.8, 1 mM
. To make 1 litre of 10× column
buffer, weigh out 151.2 g of PIPES, 3.8g of EGTA, and
2 ms of a 1M
stock solution of magnesium chloride and
add 45.6g potassium hydroxide. This will adjust the
pH to about pH 6.7 and further adjustments can be
made using a 10M
solution of potassium hydroxide.
The BRB80 conversion buffer is added to the purified
tubulin that has been eluted from the phosphocellulose
column to convert the buffer from column
buffer composition to BRB80 prior to storage of the
tubulin. The BRB80 conversion buffer is added in a
ratio of I part conversion buffer to 20 parts tubulin in
To make up 150mls of BRB80 conversion buffer
combine 47.6g PIPES, 4.2ml MgCl2
), and 1.25ml
). This should then be brought to pH 6.8
with potassium hydroxide. Having the correct pH is
Nucleotides are made as stock solutions as 100mM
ATP and 200mM
GTP. It is important that the pH of
these nucleotides does not become acidic, as hydrolysis
will result. Consequently, they are made up in
water and adjusted to pH 7.5 with sodium hydroxide.
The ATP solution is made 200mM
with respect to
. This is not the case with the GTP solution, as
this will cause precipitation. These solutions should be
stored at -80°C until needed.
Protein concentration determinations are made
using the Bio-Rad version of the Bradford protein
assay, reading the absorption at 595 nm using bovine
serum albumin as a reference standard.
: All g forces quoted are Rmax
Because brain tissue has a high density of microtubules
in the dendrites and axons of its nerve cells,
this is the tissue of choice for a tubulin preparation.
Porcine brain is readily available from slaughterhouses.
It is essential, however, to get the brains as fresh as possible, as protein degradation will begin to
take place soon after death. The brains should be warm
when they are received and should be plunged immediately
into a mixture of ice and saline solution for
transport back to the laboratory. Do not use cold
1. Preparation of Brain Tissue for the First
Cautionary note: As alluded to earlier, possible
contact with pathogens associated with nervous tissue
should be minimized by always handling the brains
with gloves. Waste tissue should be disposed of by
At the laboratory cold room the brains should be
stripped of brain stems, blood clots, and meninges
(kitchen paper tissue is very good for this purpose).
The brains are then weighed, transferred to the
Waring blender, and cold BB containing 0.1% β-
mercaptoethanol and 1 mM
ATP, but without any protease
inhibitors, is added in the ratio of 1 litre of buffer
per kilogram of brain tissue. The brains are then
homogenized twice for approximately 30s each. Typically
it can be expected to need approximately 5 litres
of BB for 30 porcine (approximately 12 bovine) brains
giving about 10 litres of total homogenate. If a large
capacity rotor is available, this homogenate can be
poured directly into the 1 litre centrifuge bottles and
then centrifuged at 12,000g for 15min. Using this
option has the advantage that any unhomogenized
tissue and air (from the blending procedure) are
removed rapidly. The homogenate is centrifuged
further at 34,200g
for 60min (SLA1500) at 4°C.
2. First Warm Centrifugation
3. Second Cold Centrifugation
- Pool and pour the supernatants (approximately
5 litres for a 30 porcine brain preparation) into two 5-
litre Ehrlenmeyer flasks.
- Add a half volume of anhydrous glycerol that
has been prewarmed to 37°C and then adjust the GTP,
ATP, and MgCl2 concentrations to 0.5, 1.5, and 3 mM,
respectively. It is essential to bring the tubulin solution
above 30°C as quickly as possible.
- Warm the tubulin to 30°C under a continuously
flowing hot tap while swirling the flask. At some point
after the tubulin has reached 30°C it will be noticed
that the solution becomes noticeably more viscous,
indicating that polymerization is beginning to take
- Incubate the tubulin for a further 60min at 37°C in a water bath before recovery by centrifugation at
50,000 g for 150 min at 37°C in Beckman type 19 rotors.
4. Second Warm Centrifugation
- Resuspend the pellets in approximately 700 ml of
PB (with 0.1% β-mercaptoethanol) at 4°C. using either
a Dounce or a continuous flow homogenizer. At this
point, determine the protein concentration of the
resuspended pellet solution and lower the concentration
to 25mg/ml by the addition of cold PB.
- Leave the solution on ice for 40min to allow
for complete depolymerization before centrifuging at
150,000g for 30min in Beckman type 45Ti rotors at 4°C.
5. Third Cold Centrifugation
- Pool the supernatants from this centrifugation
and adjust the concentrations of ATP, GTP, and MgCl2 to 1, 0.5, and 4 mM, respectively.
- As before, add a half volume of anhydrous glycerol
prewarmed to 37°C and warm the entire mixture
to 37°C. Allow the tubulin to polymerize at this temperature
for a further 40min prior to centrifugation at
150,000g at 37°C for 1 h in Beckman type 45Ti rotors.
If time is short, the polymerized tubulin pellets can be
frozen in liquid nitrogen and stored at -80°C.
6. Purification of Tubulin over a Phosphocellulose
- Collect and resuspend the polymerized tubulin
pellets in a total volume of 200ml of PB with 0.1%
β-mercaptoethanol using either the continuous flow or
the Dounce homogenizer.
- Determine the protein concentration and dilute
the solution with PB as necessary to a final concentration
of 35 mg/ml.
- Allow the tubulin to depolymerize on ice for
a further 40min and then centrifuge at 150,000g for
30 min in the Beckman type 45Ti rotor.
- The supernatant is now ready for loading onto
the phosphocellulose column to separate the tubulin
from microtubule-associated proteins.
B. Cycling Tubulin
- Load the depolymerised tubulin onto a 1-1itresized
column (at 4°C) that has been equilibrated previously
in CB. Tubulin should be loaded onto the
column at a flow rate of approximately 1.5ml/min.
Once loaded, however, the flow rate can be increased
to 6ml/min or even faster if the phosphocellulose
shows little sign of compressing, although some compression
- The purified tubulin elutes in the flow through.
Pool the protein peak and determine the protein concentration.
The purified tubulin in CB can then be
snap-frozen in liquid nitrogen or converted to tubulin
in BRB80 using BRB80 conversion buffer prior to freezing and subsequent storage at -80°C. When run on a
12% polyacrylamide gel, the purified tubulin, when
stained with Coomassie, should appear as a single
band free of any trace of contaminating proteins. The
MAPs are retained on the column and these proteins
can be eluted using CB containing 1M NaCl.
It is inevitable that some tubulin will become inactivated
or denatured during passage through the phosphocellulose
column, as tubulin is an unstable entity.
It is therefore recommended to "cycle" the tubulin
after it passes through the column. To cycle tubulin
means that it is polymerised at 37°C, reisolated by centrifugation
through a glycerol cushion, and depolymerised
at 4°C prior to freezing. This can be performed
either immediately after elution from the column or
after it has been stored at -80°C in CB. In both cases
the tubulin buffer should be converted to BRB80 as
described earlier. There are several advantages to
cycling the tubulin: it enriches for active tubulin
dimers, removes free nucleotides, removes denatured
tubulin and other impurities, and concentrates the
tubulin to approximately 10-20mg/ml (cf. 5-10mg/
ml as it comes through the column.). The resulting
cycled tubulin is suitable for use in in vitro
as video microscopy (e.g., Andersen et al.
, 1994). It is
also recommended to use cycled tubulin for biochemical
studies (e.g., purification of MAPs) because it is
much easier to control the amount of microtubules
formed when this highly active tubulin is used compared
to that obtained directly from the phosphocellulose
To obtain a large stock of identical cycled tubulin,
it is recommended to cycle approximately 30ml of
tubulin obtained directly from the phosphocellulose
C. Preparation of Phosphocellulose Column
- Prewarm the rotor (a Beckman Ti50, TLA100, or
MLA 80 rotor is suitable) in a water bath at 37°C. Fill
the rotor tubes to half their volume with a glycerol
cushion consisting of 60% glycerol in BRB80 (no GTP),
place these in the rotor, and allow them to equilibrate
to 37°C. While this is happening, proceed with polymerisation
of the tubulin.
- Thaw the tubulin rapidly using a water bath set
at 37°C until the tube is half full of ice and then continue
to thaw the remainder on ice. Adjust the solutes
to glycerol PB using anhydrous (100%) glycerol. Allow
polymerisation to occur for 40min at 37°C.
- Layer the polymerised tubulin onto the prewarmed
cushions using tips with large openings to
avoid depolymerising the microtubules. Centrifuge at
226,240g (50,000rpm in the Beckman Ti50 rotor) for
60min or, alternatively, at 70,000rpm for 30min in the
TLA100 rotor at 37°C.
- Aspirate away the supernatant above the
cushion. Rinse the cushion interface twice with water.
Aspirate away the cushion. Resuspend the pellet in
0.25x BRB80 + 0.1% β-mercaptoethanol on ice using an
homogeniser to depolymerise the microtubules. The
volume of buffer used for the resuspension is chosen
so that the final tubulin concentration is between 10
and 20mg/ml (based on the assumption that approximately
half the tubulin from the phosphocellulose
column will polymerise). Incubate on ice for 15min
and then add 5x BRB80 to adjust the buffer to 1x
BRB80. (Note: The volume of 5x BRB80 to add is
3/16ths of the volume of 0.25x BRB80-tubulin.)
- Sediment the undepolymerised microtubules by
centrifuging the sample at 213,483g (70,000rpm in the
Beckman TLA100.2 rotor) for 15min at 4°C.
- Aliquots (10-200µl) of the concentrated tubulin
can be made, which should then be snap frozen in
liquid nitrogen. The tubulin can then be stored either
in liquid nitrogen (indefinitely) or at -80°C (for at least
For large-scale preparations of tubulin (finally
giving 1 g or more of purified tubulin) a phosphocellulose
column of approximately 1 litre volume will be
necessary. For successful preparation of PC, it has to be
equilibrated for short periods
first in base and then in
acid, interspersed with water washes. This is normally
achieved by suspension of the PC in either the acid or
the base solution and then rapid filtration over a sintered
glass funnel where the PC can be washed with
large volumes of water. Large volumes of PC are,
however, quite cumbersome to handle and the importance
of setting up an efficient filtration system before
commencing the PC preparation cannot be overemphasised.
With volumes as large as 1 litre it may be
unwise to rely on running water aspirators, as the
vacuum produced may be insufficient (but for small
volumes of PC these may be suitable). It is better to use
a membrane-type pump and if, possible, to connect this
to a wide, sintered glass funnel over which Whatmann
3 MM filter paper has been placed. Alternatively, and
we have used this quite successfully, use some nylon or
poiypropylene meshing (available from SpectraMesh),
as this allows rapid filtration rates with almost no risk
of tear as there is when using Whatmann filter paper.
When considering what volume of phosphocellulose
is needed, our laboratory uses 200ml phosphocellulose
to obtain 300-600mg of purified tubulin.
When equilibrated in buffer, 1 g of PC powder will give
between 5 and 6 ml of PC column matrix (depending
on the salt concentration), but do allow for some loss
due to the removal of fines when calculating the
amount of PC to prepare.
Phosphocellulose is obtained from Whatmann Scientific
Ltd. and the column and adaptors can be
obtained from Kontes, but any column with similar
dimensions (10 x 30 x 4.8cm) should be suitable.
: All stages should be performed at 4°C.
- Having determined the amount of phosphocellulose
that is needed using the information just given,
slowly hydrate the dry powder by washing twice in
95% ethanol, once in 50% ethanol, and finally once in
- Resuspend the hydrated phosphocellulose in 25
volumes (liquid volume per original dry weight of
phosphocellulose) of 0.5 M NaOH and stir gently (stirring
too fast produces fines, which will result in slow
column flow rates if not removed later) for 5 min. Filter
the phosphocellulose rapidly to remove the NaOH
solution and continue to rinse with water until the pH
of the washings is lower than 10 (usually at least three
times the volume of the NaOH solution used).
- As soon as the pH is sufficiently low, resuspend
the phosphocellulose in 25 volumes of 0.5M HCl (i.e.,
the same volume as the NaOH solution as used earlier)
and again stir gently for 5min and filter quickly.
Continue to wash with water until no longer acid m
usually around 10 times the volume of the HCl solution
- If the column is not to be poured at this stage, the
phosphocellulose should be resuspended and stirred
for 5 min in a 2 M solution of potassium phosphate, pH
7.0. The PC can then be resuspended and stored in a
solution of 0.5M potassium phosphate, pH 7.0, containing
20 mM sodium azide as preservative. When the
phosphocellulose is removed from storage, it should
be resuspended and washed in at least 3 volumes of
- At this stage, whether the column material was
stored or not, the phosphocellulose should be resuspended
in at least 3 volumes of water and transferred
to a large measuring cylinder. The phosphocellulose
should be stirred and then allowed to settle naturally.
After the bulk of the phoshocellulose has settled, it will
be noticed that there is a cloudy layer just above it.
These are the fines and should be removed by aspiration
to ensure fast column flow rates. This cycle of
resuspension, stirring, and aspiration of the fines
should be repeated until no more fines are visible.
- Resuspend the phosphocellulose in column
buffer solution and then degas for 30min prior to
pouring the column.
- After use, wash the phosphocellulose column
extensively and replace the buffer with a 50mM potassium phosphate buffer containing 20 mM sodium
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