Tubulin Preparat

互联网2013-09-05

1065

Materials

3 - 5 Fresh Pig Brains

1 M GTP

1 M Magnesium Sulfate

PM buffer =

100 mM Pipes, pH 6.9
2 mM EGTA
1 mM Magnesium Sulfate
2 mM DTT

PM-4M Buffer =

100 mM Pipes, pH 6.9
2 mM EGTA
1 mM Magnesium Sulfate
2 mM DTT
4 M Glycerol

PM-8M Buffer =

100 mM Pipes, pH 6.9
2 mM EGTA
1 mM Magnesium Sulfate
2 mM DTT
8 M Glycerol
0.1 mM GTP

PC Column (20 mL phosphocellulose resin in 2.5 cm diameter column) equilibrated in PM buffer

Special Equipment

Waring Blender

Dounce homogenizer (15 mL)

Procedure

1.	Obtain pig brains and measure the weight. Keep on ice.

2.	Working rapidly, remove meninges and blood vessels with Kimwipes at 4°C. Prepare enough space in cold room for 2-3 persons working together at this step. (Need bench cover, trash bag, Waring blender, 300 ml beaker in ice bucket.)

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.

4.	Centrifuge the homogenate for 15 min at 4°C at 9 500 rpm in the Sorvall SS-34 rotor (6 500 g).

5.	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.

6.	Decant the supernatants into a graduated cylinder and check the volume (V1).

7.	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.

8.	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.

9.	Centrifuge the tubes for 60 min at 27°C at 30,000 rpm to pellet the assembled microtubules.

10.	At room temperature, decant the supernatants from the firm and slightly opalescent pellets.

11.	Resuspend the pellets in a volume of 0.25 x V1 of ice-cold PM buffer.

12.	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.

13.	Incubate the suspension on ice for 30 min to depolymerize microtubules.

14.	Transfer into ice-cold ultracentrifuge tubes and centrifuge at 30 000 rpm at 4°C for 60 min in a Beckman Ti70 rotor.

15.	Carefully remove the supernatants from the pellets, which are sometimes loose, and pool in a graduated cylinder. Note the volume (V2).

16.	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.

17.

A second cycle of polymerization/centrifugation and depolymerization/ centrifugation is carried out by first incubating the mixture at 34°C as described above (steps 8-10). Resuspend the resulting pellets in 0.25 x V2 of cold PM buffer, incubate at on ice for 30 min and centrifuge again (steps 11-15).

18.	The resulting cold supernatant is ~90% pure tubulin and is called twice-cycled tubulin. Further purification is by phosphocellulose column chromatography.

19.	Prepare a PC column (20 mL PC resin in 2.5 cm diameter column) and equilibrate the column in PM buffer.

20.	Prior to collection, 1 M Magnesium Sulfate is added to the collection tubes to make a final concentration of 1 mM.

21.

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.

Notes