Exercise 9.6 - Polymerization Induced Changes in Absorbance

LEVEL II

Materials

• UV spectrophotometer
  
• Water baths set at 10° C and 37° C
  
• Tubulin extract from Exercise 9.5
  
• MT buffer, ATP and GTP

Procedure

1. Turn on the spectrophotometer and adjust the wavelength to 350 nm. Adjust the spectrophotometer to read absorbance, using microtubule buffer as a blank.

2. Starting with the tubulin extract in the water bath and equilibrated to 10° C, add ATP and GTP as in steps 1 and 2 of Exercise 9.5. Immediately measure the absorbance of the solution. Speed is important! Place the sample still in the spectrophotometer cuvette back into the 10° C waterbath.

3. At 30 second intervals remove the cuvette, wipe it dry and measure the absorbance. Immediately replace the sample and cuvette back in the 10° C waterbath. Repeat this for a total period of 3 minutes.

4. After the 3 minute interval, place the cuvette and its contents in the 37° C waterbath.

5. Continue to read the absorbance at 30 second intervals, returning the sample to the 37° C waterbath between readings.

6. When the absorbance value stabilizes (about 5 minutes), replace the sample and cuvette back in the 10° C bath and continue to measure the absorbance at 30 second intervals.

7. Plot absorbance against time, indicating points of temperature change.

Notes

You may monitor the kinetics of tubule formation by continuously measuring the absorbance of the solution at 350 nm, provided a temperature controlled sample holder is available. Preferably, the sample temperature should be able to be rapidly changed from 10° C to 37° C and back. This can be accomplished via a temperature-controlled kinetic spectrophotometer (such as a Beckman DU Kinetic spectrophotometer, or equivalent). Alternatively, a water jacketed sample cuvette can be engineered with some simple tubing and valves to switch the water flow through the jacket.

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