Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis.
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.
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas.|Department of Biochemistry and Molecular Biology, Gannan Medical University, Ganzhou, Jiangxi Province, China.
Xiaoting
X
Luo
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas.
Jinzi
J
Wu
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas.
Zhen
Z
Jin
Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas.|Corresponding author (liang-jun.yan@unthsc.edu).
Liang-Jun
LJ
Yan
D043182:Carboxylesterase / Q000032:analysis; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D007202:Indicators and Reagents; D008928:Mitochondria / Q000737:chemistry*; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D013194:Staining and Labeling
D007202:Indicators and Reagents; D011506:Proteins; D008058:Dihydrolipoamide Dehydrogenase; D043182:Carboxylesterase
blue native gel; electrophoresis; esterase; mitochondria; non-gradient