过去 15 年的研究揭示了细胞外热休克蛋白90α（extracellular heat shock protein 90alpha，简称eHsp90α）在多种人类疾病的发展中起着至关重要的作用，包括创伤愈合、消瘦（肌肉消耗）、炎症性疾病和癌症。经过高纯度的eHsp90α蛋白鉴定，已确定其具有促进细胞存活和刺激细胞迁移的两个功能。然而，eHsp90α的分泌机制和分离方法尚待规范化。在众多报道的方法中，差速离心被认为是“黄金标准”，主要是因为它能够从培养细胞的条件培养基中定量回收eHsp90α。在这里，我们描述了一个修订后的方案，用于从肿瘤细胞条件培养基中分离出具有不同直径范围的三个细胞外囊泡部分和剩余的无囊泡上清液，以进行生化分析，特别是对eHsp90α进行分析。可以使用已知量的重组Hsp90α蛋白在同一SDS-PAGE上定量相对的eHsp90α含量。我们相信，这种改进后的方法将证明是研究培养细胞及其外囊泡中eHsp90α的有用工具。© 2023年作者(s)。Springer Science+Business Media, LLC的独家许可，属于Springer Nature的一部分。

Studies of the past 15 years have revealed a critical role for extracellular heat shock protein 90alpha (eHsp90α) in the development of several human disorders, including wound healing, cachexia (muscle wasting), inflammatory diseases, and cancers. The two established functions of highly purified eHsp90α protein are to promote cell survival and to stimulate cell migration. However, the mechanism of secretion and the method of isolation of eHsp90α remained to be standardized. Among the half a dozen reported methodologies, differential centrifugation is considered the "gold standard" largely for its quantitative recovery of eHsp90α from a conditioned medium of cultured cells. Herein, we describe a revised protocol that isolates three fractions of extracellular vesicles with distinct ranges of diameters and the leftover vesicle-free supernatant for biochemical analyses, especially eHsp90α, from tumor cell-conditioned media. Quantitation of the relative amount of eHsp90α can be carried out with known amounts of recombinant Hsp90α protein on the same SDS-PAGE. We believe that this modified methodology will prove to be a useful tool for studying eHsp90α in cultured cells and beyond.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.