Non‐Gradient Blue Native Polyacrylamide Gel Electrophoresis

Abstract Gradient blue native polyacrylamide gel electrophoresis (BN‐PAGE) is a well established and widely used technique for activity analysis of high‐molecular‐weight proteins, protein complexes, and protein‐protein interactions. Since its inception in the early 1990s, a variety of minor modifications have been made to this gradient gel analytical method. Here we provide a major modification of the method, which we call non‐gradient BN‐PAGE. The procedure, similar to that of non‐gradient SDS‐PAGE, is simple because there is no expensive gradient maker involved. The non‐gradient BN‐PAGE protocols presented herein provide guidelines on the analysis of mitochondrial protein complexes, in particular, dihydrolipoamide dehydrogenase (DLDH) and those in the electron transport chain. Protocols for the analysis of blood esterases or mitochondrial esterases are also presented. The non‐gradient BN‐PAGE method may be tailored for analysis of specific proteins according to their molecular weight regardless of whether the target proteins are hydrophobic or hydrophilic. © 2017 by John Wiley & Sons, Inc.