TITLE: In vitro preparation of single filamentous microtubules optimized for dynamic and electrophoretic light scattering measurements.
AUTHORS: Annitta George, Ernesto Alva, Lorenzo Brancaleon, Marcelo Marucho

[DESCRIPTION]
The lack of essential information on sample preparation and the need to characterize and understand microtubules (MTs) in various biological functionalities within the cell have led us to develop a high-quality polymerization protocol to prepare fully formed MTs in vitro, which is particularly designed for Dynamic light scattering (DLS) and Electrophoretic light scattering (ELS) experiments for further analysis. This protocol details the reconstitution of tubulin, GTP, and Taxol, the preparation of PEM, cushion buffer, and polymerization buffers, the step-by-step polymerization process, and the use of Taxol to stabilize the microtubule filaments without precipitation. This polymerization protocol successfully generated samples for light scattering experiments using buffers that replicate physiological conditions. This has provided consistency in preparing stable, diluted, aggregate-free, homogenous microtubule filament samples that could benefit many other scientific research groups currently working in the field. Additionally, it can easily be adapted to prepare samples using other buffers and biological fluids.

Graphical abstract:


 
This sample preparation protocol requires 3 days.
All the buffers and solutions are prepared to make enough of four samples of fully developed microtubules in 100 millimolar concentration of potassium chloride (KCl), with a total volume of 1 milliliter of solution. (See Materials sections).

[STEPS]
SECTION: First-day procedure: Stock preparations
1. 
 

SECTION: First-day procedure: Stock preparations
2. 100 mM PIPES:

Add 302.4 mg of PIPES to a 15 mL polypropylene centrifuge tube.

SECTION: First-day procedure: Stock preparations
2.1. Add 13-15 drops of 5 Molarity (M) KOH solution to the tube.

SECTION: First-day procedure: Stock preparations
2.2. Vortex the solution for 35 s–45 s at low to medium speed (4/10) until there is no residue.

SECTION: First-day procedure: Stock preparations
2.3. Add 9.3 mL -9.5 mL of H2O ultra-pure to the tube.

SECTION: First-day procedure: Stock preparations
2.4. Vortex the solution for 35 s–45 s at low to medium speed (4/10).

SECTION: First-day procedure: Stock preparations
3. 10 mM EGTA:

Add 38.04 mg of EGTA to a 15 mL polypropylene centrifuge tube.

SECTION: First-day procedure: Stock preparations
3.1. Add 1-2 drops of 5 Molarity (M) KOH solution to the tube.

SECTION: First-day procedure: Stock preparations
3.2. Vortex the solution for 35 s–45 s at low to medium speed (4/10) until there is no residue.

SECTION: First-day procedure: Stock preparations
3.3. Add 9.6 mL-9.7 mL of H2O ultra-pure to the tube.

SECTION: First-day procedure: Stock preparations
3.4. Vortex the solution for35 s–45 s at low to medium speed (4/10).

SECTION: First-day procedure: Stock preparations
4. 50 mM MgCl2 : 

Add 47.61 mg of MgCl2 to a 15 mL polypropylene centrifuge tube.

SECTION: First-day procedure: Stock preparations
4.1. Add 10 mLof H2O ultra-pure to the tube.

SECTION: First-day procedure: Stock preparations
4.2. Vortex the solution for 35 s–45 s at low to medium speed (4/10).

SECTION: First-day procedure: Stock preparations
5. 117.47 mM KCl:

Add 87.52 mgof KCl to a 50 mL polypropylene centrifuge tube.

SECTION: First-day procedure: Stock preparations
5.1. Add 10 mLof H2O ultra-pure to the tube.

SECTION: First-day procedure: Stock preparations
5.2. Vortex the solution for 35 s–45 sat low to medium speed (4/10).

 


 

SECTION: Second-day procedure: Tubulin buffers preparations
6.   
 


 


SECTION: Second-day procedure: Tubulin buffers preparations
7. PEM or BRB80 or general tubulin buffers (pH 6.91):

Add4.8 mLof PIPES to a 50 mL polypropylene centrifuge tube.
SECTION: Second-day procedure: Tubulin buffers preparations
7.1. Add 240 µL of MgCl2 to the tube.
SECTION: Second-day procedure: Tubulin buffers preparations
7.2. Add 300 µL of EGTA to the tube.

SECTION: Second-day procedure: Tubulin buffers preparations
7.3. Add 660 µL of H2O ultra-pure to the tube.

7.4. Vortex the solution for 35 s–45 sat low to medium speed (4/10).

8. PEM with glycerol or cushion buffer or tubulin glycerol buffer (pH 6.97):

Add 3.2 mLof PIPES to a 50 mL polypropylene centrifuge tube.

8.1. Add 160 µL of MgCl2 to the tube.

8.2. Add200 µL of EGTA to the tube.

8.3. Add 440 µL of H2O ultra-pure to the tube.
8.4. Add 6 mL of glycerol to the mixture.

8.5. Vortex the solution for 35 s–45 s at low to medium speed (4/10).

 

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
9. Preparations:

Before proceeding with the polymerization buffers and the step-by-step procedure, one must ensure to have enough reconstituted GTP, Taxol, and protein. If you already have these, skip to the ‘Tubulin polymerization’ steps. If not, follow the procedure given below.
SECTION: Third-day procedure: Polymerization process and suspension buffer
10. a. GTP reconstitution

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
10.1. Briefly centrifuge to collect the white powder at the bottom of the storage tube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
10.2. Add 100 µL of ice-cold distilled water for a 100 millimolar (mM) stock solution.

SECTION: Third-day procedure: Polymerization process and suspension buffer
10.3. Aliquot the GTP into experiment-sized amounts as needed.

SECTION: Third-day procedure: Polymerization process and suspension buffer
10.4. Snap-freeze the GTP with liquid Nitrogen. 

SECTION: Third-day procedure: Polymerization process and suspension buffer
10.5. Store at or below -20 °C.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11. b. Taxol reconstitution

Briefly centrifuge to collect the white powder at the bottom of the storage tube.
SECTION: Third-day procedure: Polymerization process and suspension buffer
11.1. Add 100 µL Dimethyl sulfoxide (DMSO) for a 2 millimolar (mM) stock solution. 

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.2. From 2 millimolar (mM)stock solution, pipette 25 µL to a cryotube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.3. Add 475 µL of DMSO to the cryotube to make a total of 500 µLof reconstituted Taxol with 100 micromolar (µM)concentration.
SECTION: Third-day procedure: Polymerization process and suspension buffer
11.4. For 200 micromolar (µM), add 50 µL of Taxol (2 millimolar (mM)) to 450 µLof DMSO.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.5. For 100 micromolar (µM), add 25 µL of Taxol (2 millimolar (mM)) to 475 µL of DMSO.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.6. For 50 micromolar (µM), add 125 µL of Taxol (200 micromolar (µM)) to 375 µL of DMSO.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.7. For 10 micromolar (µM), add 50 µL of Taxol (100 micromolar (µM)) to 450 µL of DMSO.

SECTION: Third-day procedure: Polymerization process and suspension buffer
11.8. Store at or below -20 °C.

SECTION: Third-day procedure: Polymerization process and suspension buffer
12. c. Protein reconstitution

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
12.1. Transfer the protein powder into a 3 mL glass vial. 
SECTION: Third-day procedure: Polymerization process and suspension buffer
12.2. Add 100 µL of ultra-pure water into the glass vial containing the protein powder to reconstitute at 10 mg/mL tubulin density. 
SECTION: Third-day procedure: Polymerization process and suspension buffer
12.3. Tubulin proteins may concentrate at the solution’s surface or walls of the vial. Vortex the solution for 30 s–40 s at low to medium speed (4/10) to dissolve the protein powder as much as possible. 
SECTION: Third-day procedure: Polymerization process and suspension buffer
12.4. Add 1.5 mL of PEM buffer to the protein solution in the glass vial.

SECTION: Third-day procedure: Polymerization process and suspension buffer
12.5. Vortex once more for 35 s–45 s at low to medium speed (4/10) to dissolve the aggregates as much as possible. If aggregates are still present, pipette and mix the solution to achieve a homogeneous protein solution.
SECTION: Third-day procedure: Polymerization process and suspension buffer
12.6. Aliquot the solution into experimental-sized amounts according to the number of experiments needed. Transfer the small-size solutions into cryotube vials. We recommend aliquoting the protein solutions into multiples of 400 µL(1×400 µL, 2×400 µL, 3×400 µL, …).

SECTION: Third-day procedure: Polymerization process and suspension buffer
12.7. Once the wanted protein solutions are aliquoted, snap-freeze the cryotube vials with liquid nitrogen and immediately store them at -80 °C.

SECTION: Third-day procedure: Polymerization process and suspension buffer
13. Tubulin suspension or polymerization buffer

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
13.1. Add 1 mLof PEM to a 15 mL polypropylene centrifuge tube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
13.2. Add 20 µLof reconstituted GTP to the tube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
13.3. Add 166.6 µL of PEM with glycerol to the tube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
13.4. Vortex the solution for 35 s–45 s at low to medium speed (4/10).

SECTION: Third-day procedure: Polymerization process and suspension buffer
13.5. Keep the solution on ice.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14. Tubulin polymerization

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.1. Extract one of the cryotube vials containing 1×400 µL of protein solution from the -80 °C freezer. 

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.2. De-frost the cryotube atRoom temperature for 5 min. 

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.3. Incubate the vial on ice for 60 min to depolymerize tubulin oligomers that may form during storage.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.4. Extract 100 µL from the cryotube vial and transfer to a 1.5 mL polypropylene microcentrifuge tube.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.5. Add 100 µLof polymerization buffer to the microcentrifuge tube containing the 200 µL protein solution to start polymerization.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.6. Vortex the solution for 40 s–50 s at low speed (3–4/10). 
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.7. Place the tubulin solution samples on ice for 10 min to bind GTP.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.8. Incubate the protein solution in the Grant SUB Aqua 12 Plus water bath at 37 °Cfor 50 min.

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.9. Set the 5–350 µL Pipette+ in pipette mode at very low speed (1/10) and add11 µL Taxol from 10 micromolar (µM)stock solution.
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.10. Incubate the samples for 10 min in the water bath.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.11. Set the 5–350 µL Pipette+ in titration mode at very low speed (1/10) and add20 µL Taxol from 50 micromolar (µM) stock solution.
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.12. Incubate the samples for 10 min in the water bath.

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.13. Set the 5–350 µL Pipette+ in titration mode at very low speed (1/10) and add 26 µL Taxol from 100 micromolar (µM) stock solution.
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.14. Incubate the samples for the last 15 min in the water bath to reach the stabilization of microtubules with Taxol.
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.15. Turn on the centrifuge 10 min–15 minbefore the previous step (14.14) is finalized. Set the centrifuge at 25000 rcf, 90 min, 25 °C. The acceleration and deceleration should both be set at a very low (2/10) speed. 

 

SECTION: Third-day procedure: Polymerization process and suspension buffer
14.16. Centrifuge the protein solutions for 90 min. 
SECTION: Third-day procedure: Polymerization process and suspension buffer
14.17. Both PEM-T and KCl-T are prepared during the time of centrifugation.

PEM-T preparation:

Add 212.4 µL of PEM buffer to a 1.5 mL polypropylene microcentrifuge tube.
Add 37.6 µL of Taxol from 100 micromolar (µM) stock solution.
Vortex the solution for 35 s–45 s at low to medium speed (4/10).
Keep the solution atRoom temperature.

Electrolyte (KCl-T) buffer preparation:

Add 851.2 µL of KCl of 117.47 millimolar (mM) mM of stock solution to a 1.5 mL polypropylene microcentrifuge tube.
Add 10 µL of Taxol from 50 micromolar (µM) of stock solution to the KCl solution.
Vortex the solution for 35 s–45 s at low to medium speed (4/10).
Add 30 µLof Taxol from 100 micromolar (µM) of stock solution.
Vortex the solution for 35 s–45 s at low to medium speed (4/10).
Add 50 µL of Taxol from 200 micromolar (µM) of stock solution.
Vortex the solution for 35 s–45 sat low to medium speed (4/10).
h)Keep the solutions at Room temperature.
14.18. After the centrifugation, remove the tubulin protein solution tubes from the centrifuge and place them in a styrofoam vial rack at Room temperature. 
14.19. Set the 5–350 µL Pipette+ in titration mode at a very low speed (1/10) and extract the supernatant from the solution in the following manner to avoid any possible stress in the solution that could lead to the breakage of the microtubule filaments: 100 µL, 100 µL, and 31.2 µL.
14.20. Vortex the pellet containing solution for 20 s –30 s at low speed (3–4/10). 
14.21. Incubate the pellet samples for 10 minat 37 °C.

14.22. Add 33 µL of warm PEM-T to pellet samples.
14.23. Keep the pellet samples at Room temperaturefor 10 min.
14.24. Vortex the solution for 40 s –50 sat low speed (3–4/10) to make the remaining microtubule pellet (25.8 µL) to reach the homogenous form.
14.25. Using the same Pipette+ specifications as in the previous step (14.24), add 941.2 µL of electrolyte buffer to the microtubule protein pellet (58.8 µL). 

14.26. Store the solution atRoom temperatureand leave it 50 s. 

 

14.27. The samples are ready to perform light scattering experiments.