PM Buffer: 100 mM Pipes pH 6.9, 2 mM EGTA, 1 mM Magnesium Sulfate, 2 mM DTT
PM-4M Buffer: PM buffer + 4 M Glycerol
PM-8M Buffer: PM buffer + 8 M Glycerol, 0.1 mM GTP
- Collect brains from slaughterhouse in ice cold saline.
- Working rapidly, remove meninges and blood vessels with Kimwipes at 4°C.
- Mince the tissues rapidly with scissors that have been rinsed with ethanol, then add PM-4M buffer at a ratio of 100 ml per 100 g of tissue (save a third to rinse the blender jar), and homogenize the tissue in a precooled Waring blender at the lowest speed for 40 sec. Transfer the mixture into a new beaker and rinse out the blender jar with small volume of the PM-4M buffer.
- Centrifuge the homogenate for 15 min at 4°C at 9 500 rpm in the Sorvall SS-34 rotor (6 500 g).
- Decant the supernatants into 30 ml Beckman ultracentrifuge tubes on ice and centrifuge at 30 000 rpm in a Beckman Ti70 rotor (96 000 g) for 75 min at 4°C.
- Decant the supernatants into a graduated cylinder and check the volume (V1).
- Add 1 M GTP to a final concentration of 0.5 mM and mix gently by inverting the cylinder serveral times.
- Transfer into new 30 ml ultracentrifuge tubes.
- Balance the tubes and incubate for 45 min in a water bath at 34°C (the solution at this stage is pinkish). During the incubation, warm the Ti70 rotor with tap water and set the ultracentrifuge temperature to 27°C.
- Centrifuge the tubes for 60 min at 27°C at 30,000 rpm to pellet the assembled microtubules.
- At room temperature, decant the supernatants from the firm and slightly opalescent pellets.
- Resuspend the pellets in a volume of 0.25 x V1 of ice-cold PM buffer.
- Remove the pellets from the bottoms of the tube by gently scraping with a glass rod. Pour the resulting suspension into an ice-cold Dounce homogenizer and homogenize using 5 gentle strokes of the pestle to produce a uniform suspension.
- Incubate the suspension on ice for 30 min to depolymerize microtubules.
- Transfer into ice-cold ultracentrifuge tubes and centrifuge at 30 000 rpm at 4°C for 60 min in a Beckman Ti70 rotor.
- Carefully remove the supernatants from the pellets, which are sometimes loose, and pool in a graduated cylinder. Note the volume (V2).
- Add an equal volume of PM-8M, mix gently and store at -20°C. The tubulin at this stage can be stored as long as 72 hrs. WAIT 24 HOURS BEFORE CONTINUING.
- A second cycle of polymerization/centrifugation and depolymerization/ centrifugation is carried out by first incubating the mixture at 34°C as described above (steps 9-11). Resuspend the resulting pellets in 0.25 x V2 of cold PM buffer, incubate at on ice for 30 min and centrifuge again (steps 12-16).
- The resulting cold supernatant is ~90% pure tubulin and is called twice-cycled tubulin. Further purification is by phosphocellulose column chromatography.
- Prepare a PC column (20 mL PC resin in 2.5 cm diameter column) and equilibrate the column in PM buffer.
- Prior to collection, 1 M Magnesium Sulfate is added to the collection tubes to make a final concentration of 1 mM.
- Apply the twice-cycled tubulin to the PC column and elude the column with PM buffer at a flow rate of 15 mL per hour collecting 0.5 - 1 mL fractions. The first eluate is purified tubulin protein. Do not mix with later peak fractions, which contain microtubule-associated proteins (MAPs). Pool the purified tubulin +/- MAPs, divide into 0.5 mL aliquots, freeze in liquid nitrogen and store at -80°C.