Preparation of tubulin-2
DAY 2: Cycling preparation of MT protein.
Keep the brains in an evacuated plastic ziplock bag buried in ice from the time of slaughter during transport back to the laboratory.
In a 4oC cold room, carefully and thoroughly remove the meninges and any blood-red tissue from the surface, stem, and within the folds of each brain.
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.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.
Homogenize the tissue by blending 5 s on high speed and then 45 s on low speed.
This should result in a suspension with the color and consistency of a strawberry milkshake.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).
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.Centrifuge the homogenate at 100,000 x g (29,000 rpm) for 60 min at 4oC in a 50.2Ti rotor to remove undisrupted tissue from cell cytosol.
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.
Immerse the portion of the tubes containing the cytosol in a 37oC water bath and allow MT protein to polymerize into MTs by incubating for 45 minutes.
During this polymerization incubation, warm the ultracentrifuge and 50.2Ti rotor to 25oC.Pellet MTs from the cytosol by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 25oC in a 50.2Ti rotor.
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.
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.Incubate the resuspended MTs on ice for 30 minutes with gentle mixing every 5 min to allow for MT depolymerization.
During this time chill the ultracentrifuge and 50.2Ti rotor to 4oC.Clarify the MT protein by centrifugation at 100,000 x g (29,000) rpm for 45 min at 4oC in a 50.2Ti rotor.
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.
Immerse the portion of the tubes containing the MT protein solution in a 37oC water bath and allow MTs to polymerize by incubation for 45 min.
During the incubation, warm the ultracenrtifuge and 50.2Ti rotor to 25oC.Pellet the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 25oC in a 50.2Ti rotor.
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.
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
Chill ultracentrifuge and 50.2Ti rotor to 4oC.
Prepare and equilibrate the phosphocellulose column with column buffer containing 0.5 mM GTP and 1mM DTT.
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.Thaw the MT pellets by immersing the ultracentrifuge tubes in a 37oC 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.
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.
Clarify the MT protein by centrifugation at 100,000 x g (29,000 rpm) for 45 min at 4oC in a 50.2Ti rotor.
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.
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.
Tubulin will pass through the column and come off after ~100 mls, while MT binding proteins will remain bound to the phosphocellulose resin.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).
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.
Immerse the centrifuge tubes to the level of the liquid within in a 37oC water bath and incubate for 30 min to allow MTs to polymerize. During this time, warm the 50.2Ti rotor and ultracentrifuge to 25oC.
Isolate the MTs from polymerization-incompetent tubulin by centrifugation at 100,000 x g for 30 min at 25oC in a 50.2Ti rotor.
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.
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.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 -80oC until use.
*=As published in...
Waterman-Storer, C.M. (In press) Microtubule/organelle motility assays. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. John Wiley, NY.
