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FIGURE 1.4
Polymerization activity of labeled tubulin: Tubulin from different stages of the labeling process
(5
M final concentration) in PME buffer was equilibrated at 37
C and the absorbance at
350 nm was recorded to obtain the baseline. Taxol was added to a final concentration of
5
m
M to initiate polymerization. The extent of polymerization was detected by monitoring
apparent absorption at 350 nm over time. GST-TTL was not removed from the
samples containing 3fY-tubulin.
m
Acknowledgments
We would like to thank Dr. Dan L. Sackett and Dr. Rebecca M. Kissling for
helpful scientific discussions and suggestions, Dr. Abhijit Banerjee for synthesizing 7-hydra-
zinyl-4-methylcoumarin, Mr. Daniel Wu for synthesizing 3-formyltyrosine, Mr. Robert
Ingraham and Mr. Matthew D. Antalek for purification of 3-formyltyrosine, Mr. Paul G.
Yoffe and Mr. Richard Law for assisting in standardizing this protocol, and Mr. David Tuttle
for scientific photography. This work was supported by NIH Grants GM093941 and
GM102867.
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