Facile Method of Tubulin Purification from Goat Brain for Reconstitution of Microtubule-Associated Intracellular Function.

Abstract

Tubulin/microtubule plays crucial role in eukaryotic cell division. Polymerization of αβ-tubulin heterodimers forms the microtubules, which is essential for the segregation of chromosomes during cell division and organelle positioning. Our method of tubulin purification from the goat brain includes isolation of goat brain, multiple cycles of polymerization (warming at 37°C)-depolymerization (cooling at 4°C) followed by centrifugation process. The purified tubulin from goat brain is highly functional and successfully used in different applications including reconstitution of cell like environments and understanding molecular mechanisms. Toward the end of the chapter, we have discussed, how this purified tubulin can be used for reconstitution of intracellular microtubule-associated events or function. To enable our reconstitution approach, we have developed various micropatterned-based platform and their fabrication methodology with single ligand and dual-ligand functionalizations, which are also demonstrated. These chemically functionalized micropatterned platforms are extremely useful for immobilization of tubulin/microtubule onto the localized defined area, which will be helpful in mimicking cellular phenomena like kinesin-driven transport, microtubule dynamics, etc.