Aguado Carmen, Pérez-Jiménez Eva, Lahuerta Marcos, Knecht Erwin
Intracellular Protein Degradation and Rare Diseases Laboratory, Centro de Investigación Príncipe Felipe, C./Eduardo Primo Yúfera, 3, Valencia, 46012, Spain.
Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Valencia, Spain.
Methods Mol Biol. 2016;1449:299-311. doi: 10.1007/978-1-4939-3756-1_19.
Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.
溶酶体在细胞内参与细胞器、大分子及多种底物的降解。在任何关于溶酶体在生理和病理条件下特定作用的研究中，建议采用能够实现其可重复且可靠分离的方法。然而，溶酶体的纯化是一项艰巨的任务，尤其是在培养细胞的情况下。这主要是由于这些细胞器的异质性，以及它们数量少且脆弱性高。此外，分离方法在破坏质膜的同时，必须保持溶酶体的完整性，因为其膜的破裂会释放出可能损害包括自身在内的所有细胞器的酶。以下所述的方案已在我们实验室常规用于从大鼠肝脏、NIH/3T3及其他培养细胞中特异性分离溶酶体，但也可适用于其他哺乳动物组织或细胞系。
