This method allows to generate pure assembly-competent tubulin with defined post-translational modifications in quantities sufficient to perform entire sets of in-vitro experiments. This protocol based on cycles of tubulin polymerization and de-polymerization is easy to perform in any cell biology lab. It allows to generate good amounts of tubulin with very little hands-on time.
To purify tubulin from suspension cultures, nine days before the tubulin prep, revive and amplify the desired cell type to obtain six confluent 15 centimeter dishes. Eight days before the tubulin prep, add one liter of pre-warmed medium to every spinner bottle per dish under a cell culture cabinet and place the spinners on a stirring table inside the cell culture incubator. Leave the lateral spinner caps slightly open to allow the medium to equilibrate to the atmosphere of the incubator.
The next day, use the cells from the confluent dishes to inoculate the spinner bottles. Return the spinner bottles to the incubator for one week with the lateral valves slightly open. To harvest the cells, transfer the cultures from the spinner bottles into one liter centrifuge bottles and collect cells by centrifugation.
Resuspend each pellet in 10 milliliters of ice-cold PBS, and pool the resuspended cells in a 50 milliliter tube for centrifugation at four degrees Celsius. For a 10 milliliters cell pellet, add 10 milliliters of lysis buffer to the pellet, and invert the tube several times to re-suspend the cells. To purify tubulin from adherent cell cultures, revive and amplify the desired cell type to obtain 10 confluent 15 centimeter dishes on the day of the tubulin prep.
To harvest the adherent cells, treating three dishes at a time, tilt the culture dishes to remove the medium while a second person gently washes the cells with seven milliliters of PBS-EDTA. After the washing, the second person should treat the cells with five milliliters of PBS-EDTA per dish before having a third person use a cell lifter to gently push the cells to one edge of the dish. When the cells have been detached, pool the cells from all three dishes into a 50 milliliter tube on ice and rinse each plate with an additional two milliliters of PBS-EDTA to collect any remaining cells.
After collecting the cells by centrifugation, discard the supernatant to determine the volume of the cell pellet. Add the lysis buffer, re-suspend the cells, and transfer them into a 14 milliliter round bottom tube. Sonicate the cells for approximately 45 pulses.
For mouse brain lysis, add 500 microliters of licensed buffer to a single brain harvested from an adult mouse and use a tissue blender to lyse the tissue. After obtaining the lysate at a 1/100th of its volume to an equal volume of 2X Laemmli buffer and boil the solution for five minutes. Store it at minus 20 degrees Celsius until quantification.
Collect a sample this way at each step of the tubulin prep. To clarify the lysate, centrifuge the sample and use a long needle to carefully transfer the clear supernatant to a new tube, taking care to avoid the upper floating layer. After the lysate clarification, it is essential to remove the floating material in the supernatant, otherwise it could negatively affect the efficiency of tubulin purification.
To polymerize tubulin from the clarified lysate, mix the entire volume of supernatant one with a 1/200th volume of 0.2 molar GTP and half a volume of warm glycerol in an appropriately sized tube. Gently pipette the mixture, take care to avoid introducing air bubbles. Seal the tubes with parafilm and place them in a 30 degrees Celsius water bath for 20 minutes.
After centrifugation, transfer the supernatant to a new tube. To de-polymerize the microtubules present in the pellet two, resuspend the pellet in ice cold BRB 80 for five minutes on ice. Pipette the sample first with a P1000 pipette, then with a P200 pipette, every five minutes for 20 minutes on ice.
Once the sample is homogenous, centrifuge it and transfer the obtained supernatant to a new tube. To remove microtubule associated proteins from the tubulin, mix the entire volume of supernatant three with an equal volume of preheated one molar pipe S, the same volume of preheated glycerol and a 1/100th volume of 0.2 molar GTP. Incubate the mix for 20 minutes at 30 degrees Celsius before centrifuging.
Transfer the microtubule-associated protein-containing supernatant four into a new tube, the pellet contains polymerized microtubules. After performing a third polymerization and de-polymerization, the amount of harvested tubulin can be quantified by SDS page analysis. The success of the purification process can be confirmed on a Coomassie stained SDS page.
A lower than expected yield of the purified tubulin can be due to an inefficient tubulin polymerization as evidenced by lower amount of tubulin in the second, fourth and sixth pellet fractions and higher amount in the second, fourth and sixth supernatant fractions. To quantify the purified tubulin, the samples can be run next to known quantities of bovine serum albumin. Quantitative densitometry of the protein bands then allows to calculate the quantity of purified tubulin.
The modification status of the tubulin can be confirmed by immuno blot analysis of the samples. Our protocol allows the isolation of tubulin with controlled post-translational modifications. With this tubulin, we can set up in vitro reconstitution experiments that allow us to determine the impact of tubulin modifications on micro-tubal properties, as well as on their interactions with microtubule associated proteins.
