Exercise 9.6 - Polymerization Induced Changes in Absorbance
- UV spectrophotometer
- Water baths set at 10° C and 37° C
- Tubulin extract from Exercise 9.5
- MT buffer, ATP and GTP
- Turn on the spectrophotometer and adjust the wavelength to
350 nm. Adjust the spectrophotometer to read absorbance, using
microtubule buffer as a blank.
- 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.
- 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.
- After the 3 minute interval, place the cuvette and its
contents in the 37° C waterbath.
- Continue to read the absorbance at 30 second intervals,
returning the sample to the 37° C waterbath between
- 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
- Plot absorbance against time, indicating points of
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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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- email@example.com