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Porcine brain tubulin preparation

互联网2013-11-13

830

实验试剂

100g P-11 cellulose phosphate fibrous cation exchanger (Whatman Inc, Clifton NJ) 6L 0.1M HCl

2L 0.1 M MgSO₄

2L 10x Column Buffer

~10 L 1x column buffer 10 M NaOH

300 ml Homogenization buffer (freshly prepared)

1L PM buffer 100 mM MgATP

1L PMG Buffer 100 mM MgGTP 1M dithiothreitol (DTT)

实验设备

Waring Blender Temperature controlled ultracentrifuge with the equivalent of a Beckman 50.2Ti rotor

At least 24 31.5 ml thick-walled polycarbonate ultracentrifuge tubes with screw caps

37℃ 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)

实验材料

3 fresh (<3 hrs after slaughter) pig brains

实验步骤

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.

3. 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.

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

5. 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.

6. Repeat the 0.1 M HCl treatment cycle until the pH of the phosphocellulose slurry is below 3.

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

8. 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.

9. 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.

10. 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.

11. 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).

12. Pour the column.

13. 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.

14. 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.

15. 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.

16. 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.

DAY 2: Cycling preparation of MT protein.

1. Keep the brains in an evacuated plastic ziplock bag buried in ice from the time of slaughter during transport back to the laboratory.

2. In a 4℃ cold room, carefully and thoroughly remove the meninges and any blood-red tissue from the surface, stem, and within the folds of each brain.

3. Do this by both picking tissue away by hand and wiping with kimwipes. The dry wipe will stick to the meninges, peeling away the deep red membrane from the pinkish grey nervous tissue underneath as the wipe is gently drawn across the surface of the brain.

4. Cut the cleaned brains into 2-3 cm2 cubes and weigh the tissue. Transfer the tissue to a Waring blender and add 0.5 ml/g freshly prepared Homogenization Buffer containing 1mM MgATP.

5. Homogenize the tissue by blending 5 s on high speed and then 45 s on low speed.

6. This should result in a suspension with the color and consistency of a strawberry milkshake.

7. Using a 50 ml serological pipette with 1/3 of the tip cut off and a pipette bulb with strong suction, transfer the brain homogenate to several 31.5 ml polycarbonate ultracentrifuge tubes, note the homogenate volume, and pair the tubes by weight (to 0.01 g).

8. Discard any extra homogenate that does not fit into a full rotor of centrifuge tubes. Tubulin is sensitive to proteases and easily denatured, and keeping extra homogenate for one hour during the following centrifugation step does not significantly increase the final yield.

9. Centrifuge the homogenate at 100,000 x g (29,000 rpm) for 60 min at 4℃ in a 50.2Ti rotor to remove undisrupted tissue from cell cytosol.

10. At room temperature, carefully collect the cytosolic supernatants from the tubes with a pipette, pool them in a graduated cylinder, dilute the total 1:1 with PMG, and add MgGTP to 0.2 mM to promote MT polymerization. Disburse into 31.5 ml ultracentrifuge tubes and pair them by weight.

11. Immerse the portion of the tubes containing the cytosol in a 37℃ water bath and allow MT protein to polymerize into MTs by incubating for 45 minutes.

12. During this polymerization incubation, warm the ultracentrifuge and 50.2Ti rotor to 25℃.

13. Pellet MTs from the cytosol by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 25℃ in a 50.2Ti rotor.

14. In the cold room, discard the supernatant and resuspend the MT pellet in 1/5 the volume of homogenate in PM buffer containing 0.2 mM GTP.

15. To do this, put a few mls of resuspension buffer into each centrifuge tube, as well as into a small glass Dounce homogenizer (on ice). Scrape out the sticky pellets with a round-ended weighing spatula and transfer them into the buffer in the homogenizer. Resuspend any pellet remaining in the ultracentrifuge tubes by trituration, and transfer it to the homogenizer. Homogenize the pellets by 5-10 passes with a type "A" pestle. Disburse the resuspended MTs into 31.5 ml ultracentrifuge tubes and pair them by weight.

16. Incubate the resuspended MTs on ice for 30 minutes with gentle mixing every 5 min to allow for MT depolymerization.

17. During this time chill the ultracentrifuge and 50.2Ti rotor to 4℃.

18. Clarify the MT protein by centrifugation at 100,000 x g (29,000) rpm for 45 min at 4℃ in a 50.2Ti rotor.

19. At room temperature, collect the supernatant containing the MT protein, dilute it 1:1 with PMG, add GTP to 0.2 mM, disburse it into 31.5 ml centrifuge tubes, and pair them by weight.

20. Immerse the portion of the tubes containing the MT protein solution in a 37℃ water bath and allow MTs to polymerize by incubation for 45 min.

21. During the incubation, warm the ultracenrtifuge and 50.2Ti rotor to 25℃.

22. Pellet the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 25℃ in a 50.2Ti rotor.

23. In the cold room, discard the supernatant. Add 1ml of column buffer containing 0.5 mM GTP to each tube, and being careful that the buffer is covering the pellet, immerse the ultracentrifuge tubes into liquid nitrogen to freeze the pellets.

24. Store the tubes at -80oC until the next day or whenever phosphocellulose column purification of tubulin is to be carried out.

DAY 3: Phosphocellulose column purification of tubulin from MT protein

1. Chill ultracentrifuge and 50.2Ti rotor to 4℃.

2. Prepare and equilibrate the phosphocellulose column with column buffer containing 0.5 mM GTP and 1mM DTT.

3. Turn off the peristaltic pump, and if the resin bed has settled, carefully loosen the seals on the adjuster plunger, slowly insert the adjuster plunger further until it barely touches the top of the resin, and retighten the seals. Switch the inlet tube from the reservoir of 1x column buffer to a 1L reservoir of 1x column buffer containing 0.5 mM MgGTP and 1 mM DTT, being careful not to introduce any bubbles into the inlet tube. Turn the peristaltic pump back on and adjust the speed to 1.8 ml/min. Allow at least 300 ml of buffer to be drawn through the column before loading the MT protein. During the phosphocellulose column equilibration, perform steps 3 and 4 below.

4. Thaw the MT pellets by immersing the ultracentrifuge tubes in a 37℃ water bath until the pellets turn from chalky white to completely translucent white. As soon as the pellets are thawed, place the tubes on ice immediately and take them into the cold room.

5. Resuspend the pellets on ice with a Dounce glass homogenizer and type "A" pestle (as in step 10 of preparation of MT protein above) in ~3x their volume of 1x column buffer containing 0.2 mM GTP. Transfer the resuspended MTs to a 31.5 ml ultracentrifuge tube. Allow the MTs to depolymerize by incubation with gentle mixing every 5 min on ice for 30 min.

6. Clarify the MT protein by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 4℃ in a 50.2Ti rotor.

7. In the cold room, collect the clarified MT protein supernatant and add MgGTP to a final concentration of 0.5 mM (an additional 0.3 mM) and DTT to 1mM.

8. After column equilibration, switch the column inlet tube from the buffer reservoir to the clarified MT protein. Load the MT protein onto the column at 1.8 ml/min. When all of the MT protein is loaded, switch the inlet back to the buffer reservoir and begin to collect 10 ml fractions.

9. Tubulin will pass through the column and come off after ~100 mls, while MT binding proteins will remain bound to the phosphocellulose resin.

10. Monitor the elution of tubulin either with the UV monitor by absorption at 280 nm or by adding 100 ul of each aliquot to 1 ml of freshly prepared Bradford reagent (Biorad) and looking for blue color. As each aliquot comes off, add MgGTP to a final concentration of 1 mM (an additional 0.5 mM). Pool fractions containing tubulin (usually ~100 ml).

11. At room temperature, add 0.186 g/ml glutamic acid (sodium salt) to the tubulin solution and stir slowly until it is dissolved. Disburse the solution into 31.5 ml ultracentrifuge tubes and pair them by weight.

12. Immerse the centrifuge tubes to the level of the liquid within in a 37℃ water bath and incubate for 30 min to allow MTs to polymerize. During this time, warm the 50.2Ti rotor and ultracentrifuge to 25℃.

13. Isolate the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g for 30 min at 25℃ in a 50.2Ti rotor.

14. In the cold room, resuspend the MT pellets on ice with a Dounce glass homogenizer as above, in 3x their volume of PM buffer containing 0.5 mM MgGTP. Incubate the resuspended MTs on ice for 30 min to allow MT depolymerization.

15. During this time, determine the tubulin concentration by measuring the absorbance at 280 nm (be sure to blank the spectrophotometer against the same PM buffer containing 0.5 mM MgGTP) using the extinction coefficient of tubulin = 115,000 Mol/cm ([tubulin] = (A280 x dilution factor)/extinction coefficient)). Add PM buffer containing 0.5 mM MgGTP to adjust the final protein concentration to 45 uM.

16. Disburse into several 1 ml aliquots (stock aliquots) and several 50ul aliquots (to be used directly in the MT/Organelle motility assays), freeze by immersion in liquid nitrogen, and store at -80℃ until use.

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