Derdak Zoltan, Garcia Tamako A, Baffy Gyorgy
Division of Gastroenterology & Liver Research Center, Brown Medical School and Rhode Island Hospital, Providence, RI, USA.
Methods Mol Biol. 2009;559:205-17. doi: 10.1007/978-1-60327-017-5_15.
There is an increasing evidence that uncoupling protein-2 (UCP2), a recently identified molecular sensor and suppressor of mitochondrial reactive oxygen species (ROS), plays an important role in -regulating apoptosis in different cell systems. A great technical difficulty that many groups have encountered is the reliable detection of endogenously or exogenously expressed UCP2 protein. The goal of this -chapter is to introduce the reader to techniques that we have successfully used over the years to detect UCP2 protein in various mouse and human specimens. These techniques include mitochondrial isolation and submitochondrial fractionation followed by Western blotting and UCP2 immunohistochemistry. We find that sample preparation is a key to success and it allows one to produce relevant and important data using commercially available antibodies.
越来越多的证据表明，解偶联蛋白2（UCP2）是一种最近发现的线粒体活性氧（ROS）分子传感器和抑制剂，在调节不同细胞系统中的细胞凋亡中起重要作用。许多研究小组遇到的一个重大技术难题是可靠检测内源性或外源性表达的UCP2蛋白。本章的目的是向读者介绍我们多年来成功用于检测各种小鼠和人类标本中UCP2蛋白的技术。这些技术包括线粒体分离和亚线粒体分级分离，随后进行蛋白质免疫印迹和UCP2免疫组织化学。我们发现样品制备是成功的关键，它使人们能够使用市售抗体产生相关且重要的数据。
