Purification of Bovine Brian Tubulins

Level of difficulty: High

Corresponding chapter(s) in the textbook: Chapter 14 (and 4)

Review the following terms before working on the problem: cytoskeleton, microtubules, tubulins, agarose, column chromatography, polyacrylamide gel electrophoresis, Coomassie Blue staining, Western blotting

Experiment

Tubulin was isolated from bovine brain, purified by phosphocellulose ion exchange chromatography, and further fractionated by affinity chromatography on an anti-2-tubulin-agarose column. (Note: Affinity chromatography is a powerful separation technique based on the specific, noncovalent interaction between two molecules; in this experiment: between β2-tubulin and its antibody. The antibody is immobilized on agarose beads, and the protein solution is passed through the antibody-agarose column. Unbound molecules are washed out with excess sample buffer, and the specifically bound proteins are eluted with a high-salt solution.)

The original tubulin (PC, for phosphocellulose-purified), the unbound fraction (U), and a bound fraction eluted from the column (B) were fractionated by polyacrylamide gel electrophoresis. The gels were subjected to the following procedures:

(a) Coomassie Blue protein staining

(b) Western blot with anti-2-tubulin

(c) Western blot with anti-1-tubulin

Figure

Source: Banerjee, A., M. C. Roach, P. Treka, R. F. Luduena. 1990. Increased microtubule assembly in bovine brain

tubulin lacking the type III isotype of β-tubulin. J. Biol. Chem. 265: 1794–1799.

Questions

1. Briefly describe ion exchange chromatography. What can we learn in panel a lane 1 about the molar ratio of α- and β-tubulins in the phosphocellulose-purified sample? Explain your answer.

2. What conclusion can be drawn from comparing samples 1 and 2?

3. What conclusion can be drawn from comparing samples 1 and 3?

4. Evaluate the specificity of the anti-2-tubulin antibody used in the experiment.

5. Define affinity chromatography then Evaluate the specificity of the affinity chromatography step. Was it a good way to separate the target proteins? Why or why not?

edits by Diane Vorbroker

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