A standard procedure in purifying tubulin is to extract the tissue on ice, then

Question

A standard procedure in purifying tubulin is to extract the tissue on ice, then add GTP and warm the tissue extract to 37°C. The warmed extract is then centrifuged at a speed insufficient to pellet average sized proteins, but sufficient to pellet large protein complexes. After centrifugation, the supernatant is discarded, and the pellet is redissolved in cold buffer. GTP is again added to the redissolved pellet proteins, and the solution is again warmed for a few minutes. Then centrifugation at the same speed described above is again performed. This process is repeated several times.





A. How would this process result in purification of tubulin?











































B. What contaminating proteins would likely co-purify with the tubulin?

Explanation / Answer

This process would result in the purification of tubulin by stepwise pelleting and suspension of large protein complexes such as tubulin while discarding any smaller protein complexes via the supernatant. With each consecutive centrifugation, small protein complexes are removed from the tissue leaving larger protein complexes to be compacted into a tight pellet for resuspension later. It works increasingly well because of the added effect of the property of tubulin to bind to GTP as a GTPase which makes it hold tightly to the GTP and effectively become larger and able to be spun down while as other molecules refuse to bind and become suspended in the supernatant. Contamninating proteins that would likely co purify with tubulin would be any other large globular protein complexes similar to tubulin such as immunoglobins, hemoglobins, or actin.

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