Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
Technical Contact.
STAR Protoc. 2020 Sep 18;1(2). doi: 10.1016/j.xpro.2020.100089. Epub 2020 Sep 3.
By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results. For complete details on the use and execution of this protocol, please refer to Lobo-Jarne et al. (2018) and Timón-Gómez et al. (2020).
采用带负电荷的考马斯亮蓝 G-250 染料诱导蛋白质的电荷转移，蓝色 native 聚丙烯酰胺凝胶电泳（BN-PAGE）可在保持天然构象的同时，对从细胞或亚细胞裂解物中提取的具有酶活性的多蛋白复合物进行分析。BN-PAGE 最初是为了分析形成线粒体呼吸链和 OXPHOS 系统的复合物和超复合物的大小、组成和相对丰度而开发的。在这里，我们提供了一个详细的 BN-PAGE 方案，以获得稳健和可重复的结果。有关此方案的使用和执行的完整详细信息，请参阅 Lobo-Jarne 等人。(2018 年)和 Timón-Gómez 等人。(2020 年)。
