Department of Bioscience & Bioengineering, Indian Institute of Technology Jodhpur, Karwar, Rajasthan, India.
Methods Mol Biol. 2022;2430:17-45. doi: 10.1007/978-1-0716-1983-4_2.
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.
微管/微管在真核细胞分裂中起着至关重要的作用。αβ-微管蛋白异二聚体的聚合形成微管，这对于细胞分裂过程中染色体的分离和细胞器的定位是必不可少的。我们从山羊脑提取微管蛋白的方法包括山羊脑的分离、多次聚合（在 37°C 下）-解聚（在 4°C 下冷却），然后进行离心处理。从山羊脑中纯化的微管蛋白具有高度的功能性，并成功地应用于不同的应用，包括重建类似细胞的环境和理解分子机制。在本章的最后，我们讨论了如何将这种纯化的微管蛋白用于细胞内微管相关事件或功能的重建。为了实现我们的重建方法，我们开发了各种基于微图案的平台及其制造方法，包括单配体和双配体功能化，也进行了演示。这些化学功能化的微图案化平台对于将微管蛋白/微管固定在局部定义的区域非常有用，这有助于模拟细胞现象，如驱动蛋白驱动的运输、微管动力学等。
