Preparation of Tubulin from Porcine Brain
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 microtubule motors.
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., 1991).
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 INSTRUMENTATION
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 "O" ring.
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 suppliers.
MES (M-5057), PIPES (P-6757), EGTA (E-4378), MgCl2 (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 (1.00319.2500), KH2PO4 (4873.1000), and K2HPO4 (5099. 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 (i.e., 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 EDTA, 2mM EGTA, and 0.5 MgCl2. For 1 litre, use 21.7 g MES, 4ml 0.5M EDTA stock solution, 0.1 ml 0.5M EGTA stock solution, and 0.5 ml 1 M MgCl2 stock solution.
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 0.1M PIPES, pH 6.8, 0.5 mM MgCl2, 2.0 mM EGTA, and 0.5 mM EDTA.
Approximately five litres of Column buffer (CB) is 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 EGTA, and 0.2mM MgCl2. 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 column buffer.
To make up 150mls of BRB80 conversion buffer combine 47.6g PIPES, 4.2ml MgCl2 (1M), and 1.25ml EGTA (0.2M). This should then be brought to pH 6.8 with potassium hydroxide. Having the correct pH is very important.
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 MgCl2. 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.
Note: All g forces quoted are Rmax values.
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 brains.
1. Preparation of Brain Tissue for the First Cold Spin
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 incineration.
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
4. Second Warm Centrifugation
5. Third Cold Centrifugation
6. Purification of Tubulin over a Phosphocellulose Column
B. Cycling Tubulin
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 assays such 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 column.
To obtain a large stock of identical cycled tubulin, it is recommended to cycle approximately 30ml of tubulin obtained directly from the phosphocellulose column.
C. Preparation of Phosphocellulose Column
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.
Note: All stages should be performed at 4°C.
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