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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.
Protocols
1. Preheat
Preheat a microplate reader and 96-well plate (i.e. Costar 3599) to 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 protein @ 40 mg/ml | [final] = 5 mg/ml
NOTE :
Tubulin and glycerol concentrations can be adjusted to alter polymerization kinetics.
Tubulin and glycerol concentrations can be adjusted to alter polymerization kinetics.
3. Incubate
Incubate the polymerization reaction on ice for 5 minutes. This allows the tubulin protein 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, as well as a polymerization reaction devoid of GTP to serve as a negative control.
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 pathlength correction.
Plot the absorbance readings vs. time.
