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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 Tubulin, Non-Cycled polymerization turbidity PurSolutions

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™ Cycled-Tubulin-Turbidity