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.

Recommended Products


Lyophilized Tubulin
CATALOG NO.
142001
Lyophilized Tubulin
CATALOG NO.
142001

Protocol


Steps

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 is therefore recommended to set the reader to 39°C to obtain an internal temperature of 37°C.

2. Assemble Reaction

Assemble the polymerization reaction on ice.

  • 111 µl H20 | final volume = 200 µl
  • 40 µl Tubulin PEMBuffer @ 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.

Polymerization reaction lacking GTP can be used as a negative control.

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.

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 is therefore recommended to set the reader to 39°C to obtain an internal temperature of 37°C.

Step 2

2. Assemble Reaction

Assemble the polymerization reaction on ice.

  • 111 µl H20 | final volume = 200 µl
  • 40 µl Tubulin PEMBuffer @ 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.

Polymerization reaction lacking GTP can be used as a negative control.

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.