Konovalova Svetlana
Research Programs Unit, Molecular Neurology, University of Helsinki;
J Vis Exp. 2019 Feb 12(144). doi: 10.3791/59269.
Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.
线粒体呼吸由线粒体内的氧化磷酸化（OXPHOS）复合体执行。内部和环境因素会扰乱OXPHOS复合体的组装和稳定性。本方案描述了应用蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）对培养的人类细胞中的线粒体呼吸链复合体进行分析。首先，使用洋地黄皂苷从细胞中提取线粒体，然后使用月桂基麦芽糖苷从线粒体膜中分离完整的OXPHOS复合体。接着，在带负电荷的染料考马斯蓝存在的情况下，通过梯度凝胶电泳分离OXPHOS复合体，考马斯蓝可防止蛋白质聚集并确保蛋白质复合体向阴极的电泳迁移率。最后，通过标准免疫印迹法检测OXPHOS复合体。因此，与只能研究单个OXPHOS复合体亚基的基本SDS-PAGE相比，BN-PAGE是一种方便且廉价的技术，可用于评估整个OXPHOS复合体的组装情况。
