title: A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes.
abstract: Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholate-based native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons.
authors.affiliation: Institute of Biology - Plant Physiology, Martin Luther University Halle-Wittenberg, Weinbergweg 10, D-06120 Halle/Saale, Germany.
authors.forename: Roman
authors.initials: R
authors.lastname: Ladig
authors.forename: Maik S
authors.initials: MS
authors.lastname: Sommer
authors.forename: Alexander
authors.initials: A
authors.lastname: Hahn
authors.forename: Matthias S
authors.initials: MS
authors.lastname: Leisegang
authors.forename: Dimitrios G
authors.initials: DG
authors.lastname: Papasotiriou
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ibrahim
authors.forename: Rajae
authors.initials: R
authors.lastname: Elkehal
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Volker
authors.initials: V
authors.lastname: Zickermann
authors.forename: Michael
authors.initials: M
authors.lastname: Gutensohn
authors.forename: Ulrich
authors.initials: U
authors.lastname: Brandt
authors.forename: Ralf Bernd
authors.initials: RB
authors.lastname: Klösgen
authors.forename: Enrico
authors.initials: E
authors.lastname: Schleiff
mesh_terms: D017033:Anabaena / Q000737:chemistry; D000818:Animals; D002417:Cattle; D002473:Cell Wall / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry; D000458:Cyanobacteria / Q000737:chemistry; D003840:Deoxycholic Acid / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006321:Heart; D006639:Histidine / Q000737:chemistry*; D013058:Mass Spectrometry; D000455:Medicago sativa / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008928:Mitochondria / Q000737:chemistry; D046912:Multiprotein Complexes / Q000032:analysis* / Q000302:isolation & purification; D009132:Muscles / Q000737:chemistry; D018532:Pisum sativum / Q000737:chemistry; D021381:Protein Transport; D020524:Thylakoids / Q000737:chemistry; D025062:Yarrowia / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D046912:Multiprotein Complexes; D003840:Deoxycholic Acid; D006639:Histidine