CryoBN-PAGE: A Rapid and Cost-Effective Method for the Analysis of Yeast Respiratory Complexes and Their Dynamic...

Over the last three decades, blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique that allows the resolution of large protein complexes in their native conformations, has profoundly impacted the study of mitochondrial biology and our understanding of oxidative phosphorylation (OXPHOS) system's biogenesis and organization. However, while protein samples for BN-PAGE can be prepared from permeabilized mammalian cells, the analysis of yeast OXPHOS complexes requires the prior isolation of mitochondria from whole yeast cells. This requirement limits high-throughput studies and prevents short time-course analyses. We therefore combined BN-PAGE techniques with the cryogenic milling of snap-frozen cells to develop cryoBN-PAGE, which allows for the preparation of BN-PAGE samples from whole-cell yeast lysates and avoids the cost and time associated with mitochondrial isolation. Here, we show the optimization of the method and demonstrate that it can be efficiently paired with a number of downstream applications, such as immunoblots, second-dimension SDS-PAGE, and in gel enzymatic activity assays. Furthermore, by avoiding the time-consuming mitochondrial isolation, cryoBN-PAGE allows for precise time-course assays and the investigation of rapid changes in the properties of the OXPHOS system in response to alterations in environmental conditions.