Preparationoftubulin

Although many protocols for tubulin preparation are available, the procedure described below is the simplest and highest yielding preparation I have done.  The protocol calls for 3 pig brains, and should yield ~ 60 mg of purified tubulin. 

Solutions and reagents: 
100g P-11 cellulose phosphate fibrous cation exchanger (Whatman Inc, Clifton NJ) 6L  0.1M HCl 6L  0.1 M NaOH 2L 0.1 M MgSO4 2L 10x Column Buffer ~10 L 1x column buffer 10 M NaOH 3 fresh (<3 hrs after slaughter) pig brains 300 ml Homogenization buffer (freshly prepared) 1L PM buffer 100 mM MgATP 1L PMG Buffer 100 mM MgGTP 1M dithiothreitol (DTT) Glutamic acid, sodium salt 

Equipment: 
Waring Blender Temperature controlled ultracentrifuge (Beckman L7-55) with the equivalent of a Beckman 50.2Ti rotor At least 24 31.5 ml thick-walled polycarbonate ultracentrifuge tubes (Beckman # 336091) with screw caps (Beckman # 338906) 37oC water bath 30 ml Dounce "A" glass homogenizer 2L scintered glass filter funnel and 2L sidearm Erlenmeyer flask Amicon (Danvers, MA) 44 mm x250 mm #95240 or equivalent adjustable volume column for low pressure liquid chromatography Peristaltic pump Fraction collector UV monitor or Bradford reagent (Biorad)

DAY 1:  Preparation of the phosphocellulose column.

1. Weigh out 90 g of Whatman P-11 phosphocellulose and add it to 2 L of 0.1 M NaOH in a 4 L beaker while mixing VERY gently with a glass rod.  Mix the suspension gently with the rod for 5 minutes, then allow the solid to settle for 20 min.  Aspirate off excess solution, and transfer the remaining phosphocellulose slurry to a 2L scintered glass funnel on a 2L sidearm Erlenmeyer flask  that is connected to a vacuum line.  Carefully vacuum filter the remaining 0.1 M NaOH from the phosphocellulose, BUT NEVER ALLOW THE RESIN TO RUN DRY! 
   

2. Gently scrape the phosphocellulose out of the funnel, and return it to the 4 L beaker, add 2 L of 0.1 M NaOH, mix gently for 5 min, and check the pH with pH indicator paper. 
   Repeat the mixing settling, aspiration, and filtering of the resin as in step 1. If the pH of the slurry was not above 12,  transfer the phosphocellulose to the 4 L beaker, add 2 L of 0.1M NaOH, and repeat the mixing, settling, aspiration, and filtering treatment as in step 1, leaving the wet phosphocellulose in the funnel.
   

3. Rinse 4 L of distilled water through the phosphocellulose by vacuum filtration, again, never letting the resin run dry. 
   

4. Transfer the phosphocellulose from the funnel to the 4 L beaker and add 2 L 0.1 M HCl.  Mix gently, allow the resin to settle, aspirate off excess solution, and vacuum filter the resin as in step 1. 
   Repeat the 0.1 M HCl treatment cycle until the pH of the phosphocellulose slurry is below 3.
   

5. Rinse the phosphocellulose with 4 L of distilled water by vacuum filtration. 
   

6. Transfer the phosphocellulose resin to the 4 L beaker and add 2 L of 0.1 M MgSO4.  Mix, settle, aspirate, and vacuum filter as in step 1. 
   

7. Transfer the phosphocellulose resin to the 4 L beaker, add 2 L of 10 x column buffer.  Mix gently for 10-15 min, allow the resin to settle, aspirate excess column buffer off, and vacuum filter. 
   

8. Transfer the resin to the 4 L beaker, add 2 L of 1x column buffer, mix gently for 5 min, check the pH of the slurry with a pH meter, and adjust it to 6.6 with 10 M NaOH.  Allow the resin to settle, aspirate excess buffer, and vacuum filter it. 
   Repeat the 1x column buffer treatment cycle, adjusting the pH of the slurry each time to 6.6 with 10M NaOH,  until the pH of the slurry is 6.6 without adjustment after resuspension and mixing.  Allow the resin to settle for 20 min, aspirate off buffer until the settled resin:buffer ratio is 3:1 (vol:vol).
   

9. Pour the column. 
   Mix the resin gently until it is evenly suspended in the buffer, then rapidly pour the slurry into an empty 44 mm x 250 mm liquid chromatography column (clamped to a support in the cold room) to fill it to the top.  Cover the column with parafilm, and allow the resin to settle for several hours.
   

10. Fill the column adjuster plunger with column buffer, and VERY slowly insert it into the column, being very careful not to disturb the phosphocellulose resin.  As the adjuster plunger is inserted into the column, the column inlet tubing that is attached to the plunger should be immersed in a 4L reservoir of column buffer, and should expel all air bubbles as the adjuster plunger is inserted.   Tighten and seal the adjuster plunger fittings, leaving the inlet tube in the buffer reservoir. 
    

11. Attach the outlet tubing to the peristaltic pump, and set the pump to run at 0.25 ml/min to allow the column to pack properly for the next ~48hrs while MT protein is prepared. 
    A well-packed phosphocellulose column should be perfectly even in color with no evidence of "cracks" in the resin. The better the phosphocellulose column is packed, the more concentrated the peak of elution of tubulin protein.