Microtubule Turbidity Assay

Overview


Tubulin polymerizes with characteristic kinetics including a lag phase indicative of nucleation, a log phase indicative of polymerization, and a steady-state phase indicative of no net polymer growth. These phases can be observed in real time by monitoring the turbidity of tubulin-containing solutions. The effect of microtubule associated proteins and/or pharmacological agents on microtubule polymerization kinetics can also be assessed with this assay.

For general tubulin handling instructions, please see the “Storing and Handling Lyophilized Tubulin” protocol.

Overview


Tubulin polymerizes with characteristic kinetics including a lag phase indicative of nucleation, a log phase indicative of polymerization, and a steady-state phase indicative of no net polymer growth. These phases can be observed in real time by monitoring the turbidity of tubulin-containing solutions. The effect of microtubule associated proteins and/or pharmacological agents on microtubule polymerization kinetics can also be assessed with this assay.

For general tubulin handling instructions, please see the “Storing and Handling Lyophilized Tubulin” protocol.

Recommended Products


Unlabeled Tubulin


Buffer


Protocol


1. Preheat

Preheat a microplate reader and 96-well plate (i.e. Costar 3599) to 37°C.

 NOTE :

Microplate readers often run a couple of degrees colder than expected. It may be desirable to set the reader to 39°C to obtain an internal temperature of 37°C.

2. Assemble

Assemble the polymerization reaction on ice.


  • 101 µl H20 | final volume = 200 µl
  • 40 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 2 µl DTT @ 100 mM | [final] = 1 mM
  • 2 µl GTP @ 100 mM | [final] = 1 mM
  • 30 µl glycerol @ 100% | [final] = 15%
  • 25 µl tubulin @ 40 mg/ml | [final] = 5 mg/ml

 NOTE :

Tubulin and glycerol concentrations can be adjusted to alter polymerization kinetics.

3. Incubate

Incubate the polymerization reaction on ice for 5 minutes. This allows tubulin to bind GTP.

4. Read Absorbance at 340 nm

Transfer 190 µl of the polymerization reaction to a pre-warmed 96-well plate. Add 190 µl Tubulin PEM Buffer to an additional well to serve as a blank.

Immediately collect absorbance readings at 340 nm every 30 seconds for 40 minutes.

5. Analyze

Correct the absorbance readings by subtracting the blank measurements and applying the pathlength correction for each time point.

Plot the absorbance readings vs. time.

Overview


Tubulin polymerizes with characteristic kinetics including a lag phase indicative of nucleation, a log phase indicative of polymerization, and a steady-state phase indicative of no net polymer growth. These phases can be observed in real time by monitoring the turbidity of tubulin-containing solutions. The effect of microtubule associated proteins and/or pharmacological agents on microtubule polymerization kinetics can also be assessed with this assay.

For general tubulin handling instructions, please see the “Storing and Handling Lyophilized Tubulin” protocol.

Recommended Products


Unlabeled Tubulin

Recommended Products


Recommended Products


Protocol


Step 1

1. Preheat

Preheat a microplate reader and 96-well plate (i.e. Costar 3599) to 37°C.

 NOTE :

Microplate readers often run a couple of degrees colder than expected. It may be desirable to set the reader to 39°C to obtain an internal temperature of 37°C.

Step 2

2. Assemble

Assemble the polymerization reaction on ice.


  • 101 µl H20 | final volume = 200 µl
  • 40 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 2 µl DTT @ 100 mM | [final] = 1 mM
  • 2 µl GTP @ 100 mM | [final] = 1 mM
  • 30 µl glycerol @ 100% | [final] = 15%
  • 25 µl tubulin @ 40 mg/ml | [final] = 5 mg/ml

 NOTE :

Tubulin and glycerol concentrations can be adjusted to alter polymerization kinetics.

Step 3

3. Incubate

Incubate the polymerization reaction on ice for 5 minutes. This allows tubulin to bind GTP.

Step 4

4. Read Absorbance at 340 nm

Transfer 190 µl of the polymerization reaction to a pre-warmed 96-well plate. Add 190 µl Tubulin PEM Buffer to an additional well to serve as a blank.

Immediately collect absorbance readings at 340 nm every 30 seconds for 40 minutes.

Step 5

5. Analyze

Correct the absorbance readings by subtracting the blank measurements and applying the pathlength correction for each time point.

Plot the absorbance readings vs. time.

Protocol


Step 1

1. Preheat

Preheat a microplate reader and 96-well plate (i.e. Costar 3599) to 37°C.

 NOTE :

Microplate readers often run a couple of degrees colder than expected. It may be desirable to set the reader to 39°C to obtain an internal temperature of 37°C.

Step 2

2. Assemble

Assemble the polymerization reaction on ice.


  • 101 µl H20 | final volume = 200 µl
  • 40 µl Tubulin PEM Buffer @ 5X | [final] = 1X
  • 2 µl DTT @ 100 mM | [final] = 1 mM
  • 2 µl GTP @ 100 mM | [final] = 1 mM
  • 30 µl glycerol @ 100% | [final] = 15%
  • 25 µl tubulin @ 40 mg/ml | [final] = 5 mg/ml

 NOTE :

Tubulin and glycerol concentrations can be adjusted to alter polymerization kinetics.

Step 3

3. Incubate

Incubate the polymerization reaction on ice for 5 minutes. This allows tubulin to bind GTP.

Step 4

4. Read Absorbance at 340 nm

Transfer 190 µl of the polymerization reaction to a pre-warmed 96-well plate. Add 190 µl Tubulin PEM Buffer to an additional well to serve as a blank.

Immediately collect absorbance readings at 340 nm every 30 seconds for 40 minutes.

Step 5

5. Analyze

Correct the absorbance readings by subtracting the blank measurements and applying the pathlength correction for each time point.

Plot the absorbance readings vs. time.