Viscosity Polymeriztion of Microtubules

LEVEL II

Materials

• Tubulin (Brain extract from Exercise 9.4 )
• GTP
• ATP
• Viscometer
• Waterbath or incubator at 37° C

Procedure

1. Compute the amount of GTP (M.W. 523) needed to make a final concentration 0.5 mM GTP when added to 40 ml. of brain extract in microtubule buffer. Likewise, compute the amount of dry ATP (M.W 507) needed to make a final concentration of 0.5 mM ATP.

    

2. With the brain extract in an ice bath, add both the GTP and the ATP (swirl to dissolve) and immediately transfer the mixture to a water bath at 37° C.

    

3. Use a viscometer to measure the viscosity at 3 minute intervals for a period of 30 minutes, or until change is no longer observed.

   A viscometer is a device which makes use of capillary flow. Insert a glass viscometer into a water bath at the appropriate temperature (37° C for polymerization) and add a 3.0 ml sample to the viscometer. Apply suction or pressure as appropriate to the viscometer to pull the sample into the expanded sample chamber. Release the suction (or pressure) and measure the time it takes for the sample to move from the upper mark on the viscometer to the lower mark.

   Use sucrose solutions of known viscosity to calibrate the viscometer.

    

4. Plot the change in viscosity with time of incubation.

Notes

Tubulin and MAP's will polymerize in the presence of Mg and GTP. The tubulin extract is placed in a buffer that supplies all of the necessary ingredients, except GTP. Upon the addition of GTP, the tubulin will begin to polymerize and can be monitored.