Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

2006 ◽  
Vol 27 (7) ◽  
pp. 1277-1287 ◽  
Author(s):  
Zora Nováková ◽  
Petr Man ◽  
Petr Novák ◽  
Pavel Hozák ◽  
Zdeněk Hodný
Keyword(s):  
Gel Electrophoresis ◽  
Nuclear Protein ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Blue Native

scholarly journals Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

2012 ◽  
Vol 79 (3) ◽  
pp. 974-981 ◽  
Author(s):  
Shuiquan Tang ◽  
Winnie W. M. Chan ◽  
Kelly E. Fletcher ◽  
Jana Seifert ◽  
Xiaoming Liang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Polyacrylamide Gel ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Rdase Genes ◽  
Blue Native

ABSTRACTDehalococcoides mccartyistrains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures ofDehalococcoides mccartyistrain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinctDehalococcoidesRDases and oneGeobacterRDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

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