Sep 10, 2024

Public workspaceIn vitro preparation of single filamentous microtubules optimized for dynamic and electrophoretic light scattering measurements.

DOI
dx.doi.org/10.17504/protocols.io.dm6gpzrqjlzp/v1
  • Annitta George1,
  • Ernesto Alva1,
  • Lorenzo Brancaleon1,
  • Marcelo Marucho1
  • 1Department of Physics and Astronomy, The University of Texas at San Antonio, San Antonio, USA.
Annitta George
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpzrqjlzp/v1
Protocol Citation: Annitta George, Ernesto Alva, Lorenzo Brancaleon, Marcelo Marucho 2024. In vitro preparation of single filamentous microtubules optimized for dynamic and electrophoretic light scattering measurements.. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzrqjlzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2024
Last Modified: September 10, 2024
Protocol Integer ID: 98285
Funders Acknowledgement:
NIH
Grant ID: 1SC1GM127187
Abstract
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).
Materials
Materials:

  • Laboratory Equipment/List of Equipment

  1. Analytical Explorer Pro-Scale (Ohaus Industrial Scales, model: PX84)
  2. Pipette+ (Sartorius, Andrew Alliance Stand+)
  3. Grant SUB Aqua 12 Plus water bath.
  4. Smart electronic pipettes: 5–350 µL, 10–1,000 µL, 5–10 mL (Sartorius, Andrew Alliance Stand+)
  5. Vortex mixer with standard tube head, 120V (Corning LSE, The Lab Depot, Ref:6775)
  6. Orion Star A211 pH meter, accuracy: ± 0.002 (Thermo Scientific, catalog number: X56954)
  7. Allegra 64R benchtop centrifuge machine (Beckman Coulter, product number: 367585) with an F1202 Rotor, 30,000 rpm (Beckman Coulter, catalog number: 19U313
  8. Zetasizer ULTRA, accuracy MW: ± 10% typical, temperature accuracy: 0.1 °C (Malvern Panalytical, model: ZSU5700)
  9. 2 mL glass vial (Agilent, catalog number: 20097845)
  10. Polypropylene centrifuge tube, 15 and 50 mL (Corning, catalog number: 430791)
  11. Polypropylene microfuge tube, 11 × 38 mm, 1.5 mL (Beckman Coulter, catalog number: 9080511)
  12. 12 mm square polystyrene cell cuvettes (Malvern Panalytical, number: DTS0012)
  13. 2 mL cryogenic vials (Corning, catalog number: 30721070)
  14. Business source stainless steel scissors (Fiskars, catalog number: 01-004250J)
  15. Optifit pre-sterilized tips, 10–1,000 µL (Sartorius, catalog number: PR151159)
  16. Optifit pre-sterilized tips, 5–350 μL (Sartorius, catalog number: PR149531)
  17. 5 3/4” pipets (Fisherbrand, Fisher Scientific, catalog number: 13-678-20A)
  18. Universal dip cell kit (Malvern Panalytical, catalog number: ZEN1002)

  • Biological Material

  1. Tubulin Protein (>99% Pure): Porcine Brain; store at 4 °C (Cytoskeleton, catalog number: T240)
  2. Guanosine 5’-triphosphate sodium salt (GTP); store at 4 °C (Cytoskeleton, catalog number: BST06)
  3. ReagentPaclitaxel (Taxol)Cytoskeleton Inc.Catalog #TXD01 store at 4 °C

  • Chemicals and Reagents

  1. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 67-68-5)
  2. Water for molecular biology (Millipore, catalog number: H20MB1001)
  3. Magnesium chloride, anhydrous, 99% (MgCl2) (Alfa Aesar, catalog number: W07D102)
  4. Potassium chloride (KCl) (VWR, BDH Chemicals, catalog number: 18J2256059)
  5. Potassium hydroxide (powder) (KOH) (Sigma-Aldrich, catalog number: 1310-58-3)
  6. PIPES (piperazine-N, N´-bis (2-Ethane sulfonic acid)) (Molecular biology reagent, catalog number: 194838)
  7. EGTA (ethylene glycol-bis (βaminoethyl ether)-N, N, N´, N´-tetra acetic acid) (Sigma -Aldrich, catalog number: E3889-25g)
  8. Glycerol, ACS reagent, ≥¬ 99.5% (Sigma-Aldrich, catalog number: 56-81-5)
  9. HCl volumetric standard, 0.1 N solution in water (Sigma-Aldrich, catalog number: 7647-01-0)
  10. 70% v/v denatured ethanol solution (Fisher Bioreagents, catalog number: 216731)

Buffers:

  1. Orion buffers pH 4.01, pH 7.00, and pH 10.01 (Thermo Scientific, catalog numbers: YX1, YW1, YX1)
  2. 100 mM PIPES
ABC
ReagentFinal concentrationAmount
PIPES100 mM302.4 mg
H2O ultra-puren/a9.3-9.5 mL
Total100 mM10 mL
3. 10 mM EGTA
ABC
ReagentFinal concentrationAmount
EGTA10 mM38.04 mg
H2O ultra-puren/a9.6-9.7 mL
Total10 mM10 mL
4. 50 mM MgCl2
ABC
ReagentFinal concentrationAmount
MgCl250 mM47.61 mg
H2O ultra-puren/a10 mL
Total50 mM10 mL

5. 117.47 mM KCl
ABC
ReagentFinal concentrationAmount
KCl117.47 mM87.52 mg
H2O ultra-puren/a10 mL
Total117.47 mM10 mL
6. PEM buffer (pH 7.00)
ABC
ReagentFinal concentrationAmount
PIPES80 mM4.8 mL
MgCl22 mM240 µL
EGTA0.5 mM300 µL
H2O ultra-puren/a660 µL
Totaln/a6 mL
7. Cushion buffer (pH 7.00)
ABC
ReagentFinal concentrationAmount
PIPES80 mM3.2 mL
MgCl22 mM160 µL
EGTA0.5 mM200 µL
H2O ultra-puren/a440 µL
Glycerol60 % v/v6 mL
Totaln/a10 mL
8. Polymerization buffer (pH 7.00)
ABC
ReagentFinal concentrationAmount
PEMn/a1000 µL
GTPn/a20 µL
PEM with glyceroln/a166.6 µL
Totaln/a1.186 mL
9. PEM-T
ABC
ReagentFinal concentrationAmount
PEMn/a212.4 µL
Taxol (100 µM)15 µM37.6 µL
Totaln/a250 µL
10. Electrolyte (KCl-T) buffer (pH 7.00)
ABC
ReagentFinal concentrationAmount
KCl100 mM851.2 µL
Taxol (50 µM)0.5 µM10 µL
Taxol (100 µM)3 µM30 µL
Taxol (200 µM)10 µM50 µL
Totaln/a941.2 µL





First-day procedure: Stock preparations
First-day procedure: Stock preparations
4m 30s
4m 30s

Note
  • To maintain the stock buffers biologically active, they must be remade weekly.
  • To prepare Concentration100 millimolar (mM) PIPES with a pH close to 6.8, need to add approximately 13-15 drops of Concentration5 Molarity (M) KOH solution before adding water.
  • To prepare Concentration10 millimolar (mM) GTA with pH close to 6.9, need to add approximately 1-2 drops of Concentration5 Molarity (M) KOH solution before adding water.
  • All the pipetting and titration are done using the instrument Pipette+ (Sartorius, Andrew Alliace Stand+).

100 mM PIPES:

Add Amount302.4 mg of PIPES to a 15 mL polypropylene centrifuge tube.

Add 13-15 drops of Concentration5 Molarity (M) KOH solution to the tube.

Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10) until there is no residue.

45s
Add Amount9.3 mL -Amount9.5 mL of H2O ultra-pure to the tube.

Pipetting
Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

45s
10 mM EGTA:

Add Amount38.04 mg of EGTA to a 15 mL polypropylene centrifuge tube.

Add 1-2 drops of Concentration5 Molarity (M) KOH solution to the tube.

Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10) until there is no residue.

45s
Add Amount9.6 mL -Amount9.7 mL of H2O ultra-pure to the tube.

Pipetting
Vortex the solution forDuration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

45s
50 mM MgCl2 :

Add Amount47.61 mg of MgCl2 to a 15 mL polypropylene centrifuge tube.

Add Amount10 mL of H2O ultra-pure to the tube.

Pipetting
Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

45s
117.47 mM KCl:

Add Amount87.52 mg of KCl to a 50 mL polypropylene centrifuge tube.

Add Amount10 mL of H2O ultra-pure to the tube.

Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

Note
Store all the stock buffers at Temperature4 °C .


Figure 1: Stock buffers of 100 mM PIPES, 10 mM EGTA, 50 mM MgCl2, and 117.47 mM KCl ready for refrigeration.

45s
Second-day procedure: Tubulin buffers preparations
Second-day procedure: Tubulin buffers preparations
1m 30s
1m 30s
Concentration0 Mass Percent
Note
  • Calibrate the pH meter at three-point calibration using Orion buffers Ph4.01 ,Ph7.00 , and Ph10.01 before its usage.
  • To increase/decrease the pH, add drops of a base (Concentration0.005 Molarity (M) KOH) or acid (Concentration0.1 Mass Percent HCl) solution, respectively. Add these drops carefully and constantly and check the pH, as it can rapidly change.
  • Using a 50 mL conical tube was vital since it facilitated measuring and adjusting the pH of the buffers. If the tube is smaller in size, the probe of Orion Star A211 pH meter will not be able to fit; also, there will not be enough solution to cover the tip of the probe leading to inaccurate pH readings. We recommend you consider choosing the size of conical tubes and stock buffer volume based on your pH meter probe size, as microprobes can measure smaller sample volumes effectively.
  • When adjusting the pH (PEM, and PEM with glycerol and electrolyte buffers) use glass pipettes to carefully add the base and acid solutions drop by drop into the buffers. Too many drops may significantly increase or decrease the pH, so use caution. It is important to mix well by vortexing the solutions after adding these drops; failing to do so will lead to inaccurate pH readings.


Figure 2: pH meter measuring the pH of Concentration100 millimolar (mM) KCl solution.


PEM or BRB80 or general tubulin buffers (pH 6.91):

AddAmount4.8 mL of PIPES to a 50 mL polypropylene centrifuge tube.
Pipetting
Add Amount240 µL of MgCl2 to the tube.
Pipetting
Add Amount300 µL of EGTA to the tube.

Pipetting
Add Amount660 µL of H2O ultra-pure to the tube.

Pipetting
Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

45s
PEM with glycerol or cushion buffer or tubulin glycerol buffer (pH 6.97):

Add Amount3.2 mL of PIPES to a 50 mL polypropylene centrifuge tube.

Pipetting
Add Amount160 µL of MgCl2 to the tube.

Pipetting
AddAmount200 µL of EGTA to the tube.

Pipetting
Add Amount440 µL of H2O ultra-pure to the tube.
Pipetting
Add Amount6 mL of glycerol to the mixture.

Pipetting
Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

Note
  • Titrate these solutions using the KOH solution with the pH meter to reach the normal pH of Ph7.00 and record it. A pH value of Ph6.90 to Ph7.10 is acceptable for further proceedings.
  • Store all these buffers at Temperature4 °C .

Figure 3: PEM, G-PEM, and polymerization buffer.

45s
Third-day procedure: Polymerization process and suspension buffer
Third-day procedure: Polymerization process and suspension buffer
15h 57m 20s
15h 57m 20s
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.
a. GTP reconstitution

Note
  • The lyophilized GTP (desiccated to <10% humidity) is stable for six months at Temperature4 °C .
  • The reconstituted GTP is stable if stored at or below Temperature-20 °C for six months.
  • All GTP-related steps must be performed with ice in the hood.

Briefly centrifuge to collect the white powder at the bottom of the storage tube.

Add Amount100 µL of ice-cold distilled water for a Concentration100 millimolar (mM) stock solution.

Pipetting
Aliquot the GTP into experiment-sized amounts as needed.

Snap-freeze the GTP with liquid Nitrogen.

Store at or below Temperature-20 °C .

b. Taxol reconstitution

Briefly centrifuge to collect the white powder at the bottom of the storage tube.
Add Amount100 µL Dimethyl sulfoxide (DMSO) for a Concentration2 millimolar (mM) stock solution.

Pipetting
From Concentration2 millimolar (mM) stock solution, pipette Amount25 µL to a cryotube.

Pipetting
Add Amount475 µL of DMSO to the cryotube to make a total of Amount500 µL of reconstituted Taxol with Concentration100 micromolar (µM) concentration.
Pipetting
For Concentration200 micromolar (µM) , add Amount50 µL of Taxol (Concentration2 millimolar (mM) ) to Amount450 µL of DMSO.

Pipetting
For Concentration100 micromolar (µM) , add Amount25 µL of Taxol (Concentration2 millimolar (mM) ) to Amount475 µL of DMSO.

Pipetting
For Concentration50 micromolar (µM) , add Amount125 µL of Taxol (Concentration200 micromolar (µM) ) to Amount375 µL of DMSO.

Pipetting
For Concentration10 micromolar (µM) , add Amount50 µL of Taxol (Concentration100 micromolar (µM) ) to Amount450 µL of DMSO.

Pipetting
Store at or below Temperature-20 °C .

c. Protein reconstitution

Note
  1. Tubulin is a delicate protein; to maintain the integrity and functionality of tubulin, it is crucial to handle it gently and avoid conditions that could cause damage or destabilization mainly because of mechanical stress.
  2. TheAmount1 mg tubulin protein powder needs to be extracted to begin the protein reconstitution. If the Amount1 mg of tubulin is stuck to the bottom of the vial, we suggest vortexing the protein vial at a low to medium speed (3/10) for Duration00:00:30 –Duration00:00:45 to smooth the protein and allow easy extraction when the protein is still dry.
  3. To avoid repeated thawing cycles, consider specific experimental-sized amounts.
  4. Some of the steps considered in this section are recommended by the manufacturer (Cytoskeleton, Inc.). In addition, we considered the addition of mixing steps (12.3 and 12.5), due to the rapid agglomeration/aggregation of the tubulin as the buffer is continuously added. Thus, to achieve an aggregate-free solution we implemented the following: the vortex speed is set at low to medium (4 out of 10) for a short amount of time (for Duration00:00:30 -Duration00:00:40 ). This limits the possibility of a mechanical breakdown of the protein.
  5. The aliquots containing the protein and tubulin buffers are stable for six months at Temperature-80 °C to preserve their biological activity.

Transfer the protein powder into a 3 mL glass vial.
Add Amount100 µL of ultra-pure water into the glass vial containing the protein powder to reconstitute at 10 mg/mL tubulin density.
Pipetting
Tubulin proteins may concentrate at the solution’s surface or walls of the vial. Vortex the solution for Duration00:00:30 –Duration00:00:40 at low to medium speed (4/10) to dissolve the protein powder as much as possible.
40s
Add Amount1.5 mL of PEM buffer to the protein solution in the glass vial.

Pipetting
Vortex once more for Duration00:00:35 –Duration00:00:45 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.
45s
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 Amount400 µL (1×400 µL, 2×400 µL, 3×400 µL, …).

Once the wanted protein solutions are aliquoted, snap-freeze the cryotube vials with liquid nitrogen and immediately store them at Temperature-80 °C .

Tubulin suspension or polymerization buffer

Note
  • Have ice ready in a cooler, as the polymerization buffer must be done on ice.
  • Since GTP is toxic to inhale, all steps related to GTP are performed in the fume hood with the sash lowered to ½ of its maximum opening (18 inches).

Add Amount1 mL of PEM to a 15 mL polypropylene centrifuge tube.

Pipetting
Add Amount20 µL of reconstituted GTP to the tube.

Pipetting
Add Amount166.6 µL of PEM with glycerol to the tube.

Pipetting
Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).

45s
Keep the solution TemperatureOn ice .

Tubulin polymerization

Note
  1. GTP and Taxol were initially stored at Temperature-80 °C and Temperature-20 °C respectively. When ready to prepare the solutions, thaw both solutions. We aliquoted these into experimental-sized amounts, particularly for one-time use.
  2. Following the manufacturer's recommendation, after the Duration01:30:00 centrifuge process, remove the top 90% (Amount231.2 µL ) of the supernatant from each microcentrifuge tube by following step 14.19. The amount of translucent pellets left in the microcentrifuge will be Amount25.8 µL .
  3. Before adding the reconstituted Taxol to the polymerized tubulin in the water bath, we recommend incubating them in the water bath for Duration00:05:00 to avoid thermal shock to the microtubule filaments.
  4. When homogenizing the microtubule pellet in a water bath after centrifugation (Steps 14.20 – 14.22), keep PEM-T, and KCl-T in the water bath to avoid thermal shock to the filaments.

1h 35m
Extract one of the cryotube vials containing 1×400 µL of protein solution from the Temperature-80 °C freezer.

De-frost the cryotube atTemperatureRoom temperature for Duration00:05:00 .

5m
Incubate the vial TemperatureOn ice for Duration01:00:00 to depolymerize tubulin oligomers that may form during storage.

1h
Incubation
Extract Amount100 µL from the cryotube vial and transfer to a 1.5 mL polypropylene microcentrifuge tube.

Add Amount100 µL of polymerization buffer to the microcentrifuge tube containing the Amount200 µL protein solution to start polymerization.

Vortex the solution for Duration00:00:40 –Duration00:00:50 at low speed (3–4/10).
50s
Place the tubulin solution samples TemperatureOn ice for Duration00:10:00 to bind GTP.

10m
Incubate the protein solution in the Grant SUB Aqua 12 Plus water bath at Temperature37 °C for Duration00:50:00 .

Figure 4: Four tubulin samples immersed in the Grant SUB Aqua 12 Plus water bath at Temperature37 °C for polymerization.

50m
Incubation
Set the 5–350 µL Pipette+ in pipette mode at very low speed (1/10) and addAmount11 µL Taxol from Concentration10 micromolar (µM) stock solution.
Incubate the samples for Duration00:10:00 in the water bath.

10m
Incubation
Set the 5–350 µL Pipette+ in titration mode at very low speed (1/10) and addAmount20 µL Taxol from Concentration50 micromolar (µM) stock solution.
Incubate the samples for Duration00:10:00 in the water bath.

10m
Incubation
Set the 5–350 µL Pipette+ in titration mode at very low speed (1/10) and add Amount26 µL Taxol from Concentration100 micromolar (µM) stock solution.
Incubate the samples for the last Duration00:15:00 in the water bath to reach the stabilization of microtubules with Taxol.
15m
Incubation
Turn on the centrifuge Duration00:10:00 –Duration00:15:00 before the previous step (14.14) is finalized. Set the centrifuge at Centrifigation25000 rcf, 25°C, 01:30:00 . The acceleration and deceleration should both be set at a very low (2/10) speed.

Figure 5: Microtubule samples placed in a centrifuge rotor.

1h 45m
Centrifigation
Centrifuge the protein solutions for Duration01:30:00 .
1h 30m
Centrifigation
Both PEM-T and KCl-T are prepared during the time of centrifugation.

PEM-T preparation:

  1. Add Amount212.4 µL of PEM buffer to a 1.5 mL polypropylene microcentrifuge tube.
  2. Add Amount37.6 µL of Taxol from Concentration100 micromolar (µM) stock solution.
  3. Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).
  4. Keep the solution atTemperatureRoom temperature .

Electrolyte (KCl-T) buffer preparation:

  1. Add Amount851.2 µL of KCl of Concentration117.47 millimolar (mM) mM of stock solution to a 1.5 mL polypropylene microcentrifuge tube.
  2. Add Amount10 µL of Taxol from Concentration50 micromolar (µM) of stock solution to the KCl solution.
  3. Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).
  4. Add Amount30 µL of Taxol from Concentration100 micromolar (µM) of stock solution.
  5. Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).
  6. Add Amount50 µL of Taxol from Concentration200 micromolar (µM) of stock solution.
  7. Vortex the solution for Duration00:00:35 –Duration00:00:45 at low to medium speed (4/10).
  8. h)Keep the solutions at TemperatureRoom temperature .
3m
Pipetting
After the centrifugation, remove the tubulin protein solution tubes from the centrifuge and place them in a styrofoam vial rack at TemperatureRoom temperature .
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.
Vortex the pellet containing solution for Duration00:00:20 –Duration00:00:30 at low speed (3–4/10).
30s
Incubate the pellet samples for Duration00:10:00 at Temperature37 °C .

10m
Incubation
Add Amount33 µL of warm PEM-T to pellet samples.
Pipetting
Keep the pellet samples at TemperatureRoom temperature for Duration00:10:00 .
10m
Vortex the solution for Duration00:00:40 –Duration00:00:50 at low speed (3–4/10) to make the remaining microtubule pellet (Amount25.8 µL ) to reach the homogenous form.
50s
Using the same Pipette+ specifications as in the previous step (14.24), add Amount941.2 µL of electrolyte buffer to the microtubule protein pellet (Amount58.8 µL ).

Pipetting
Store the solution atTemperatureRoom temperature and leave it DurationOvernight .

Figure 6: Microtubule samples left overnight to reach the equilibrium with the electrolyte buffer.

8h
The samples are ready to perform light scattering experiments.
Protocol references
1. E. Alva, A. George, L. Brancaleon and M. Marucho, "Hydrodynamic and Polyelectrolyte Properties of Actin Filaments: Theory and Experiments.," Polymers, vol. 14, no. 12, 6 2022.
2. E. Alva, A. George, L. Brancaleon and M. Marucho, "In vitro Preparation of Homogenous Actin Filaments for Dynamic and Electrophoretic Light Scattering Measurements," Bio-protocol, vol. 12, p. e4553, 2022.
3. J. S. Gethner and F. Gaskin, "Dynamic light scattering from solutions of microtubules.," Biophysical journal, vol. 24, no. 2, pp. 505-15, 11 1978.
4. Y. Jeune-Smith and H. Hess, "Engineering the length distribution of microtubules polymerized in vitro," Soft Matter, vol. 6, no. 8, pp. 1778-1784, 2010.
5. J. C. Lee and S. N. Timasheff, "In vitro reconstitution of calf brain microtubules: effects of solution variables.," Biochemistry, vol. 16, no. 8, pp. 1754-64, 4 1977.
6. M. L. Shelanski, F. Gaskin and C. R. Cantor, "Microtubule assembly in the absence of added nucleotides.," Proceedings of the National Academy of Sciences of the United States of America, vol. 70, no. 3, pp. 765-8, 3 1973.