MICROTUBULE TURBIDITY ASSAY
Materials:
- Lyophilized Tubulin (Cat. No. 142001) or Cycled Tubulin™ (Cat. No. 032005)
- Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, 1 mM MgCl2 , pH = 6.8)
- DTT
- GTP
- Glycerol
Equipment:
- Clear bottom 96-well plate, cell-culture treated (i.e. Costar 3599)
- Microplate reader capable of 340 nm kinetic reads at 37°C
Technical Notes:
- Avoid 384-well plates and non-treated plates
- Adjust tubulin and glycerol concentrations to alter polymerization kinetics
Protocol:
1. Pre-warm 96-well plate and microplate reader to 37°C for 30 minutes
2. Assemble the Polymerization Reactions
a. Combine DTT at 1 mM, GTP at 1 mM, glycerol at 5%, and tubulin at 5 mg/ml in Tubulin PEM Buffer
b. Incubate on ice for 5 minutes
*exclude tubulin for blank, exclude GTP for negative control
3. Polymerize and Read OD 340
a. Transfer polymerization reactions to pre-warmed 96-well plate
*avoid bubbles and work quickly
b. Immediately collect OD 340 reads at 1 minute intervals for 90 minutes
c. Subtract blank measurements and apply pathlength correction
Figure 1: Lyophilized Tubulin is polymerization-competent. Optical Density (340 nm) of Lyophilized Tubulin at 5 mg/ml in Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) supplemented with 1 mM GTP, 1 mM DTT, and 5% Glycerol at at 37°C. Distinct nucleation and polymerization phases are evident.
Lyophilized Tubulin
Figure 2: Cycled Tubulin™ is polymerization-competent. Optical Density (340 nm) of Cycled Tubulin™ at 5 mg/ml in Tubulin PEM Buffer (Cat. No. 032002; 80 mM PIPES, 1 mM EGTA, and 1 mM MgCl2, pH 6.8) supplemented with 1 mM GTP, 1 mM DTT, and 5% Glycerol at at 37°C. Distinct nucleation and polymerization phases are evident.
Cycled Tubulin™
