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Asian Journal of Biochemistry
  Year: 2006 | Volume: 1 | Issue: 4 | Page No.: 297-306
DOI: 10.3923/ajb.2006.297.306
Simultaneous Monitoring of Activities of Numerous Tricarboxylic Acid Cycle Enzymes by Blue Native Polyacrylamide Gel Electrophoresis
Ranji Singh , Robert Hamel , Daniel Chenier , Ryan Mailloux , Robin Beriault , Jeffrey Middaugh and Vasu D. Appanna

Abstract:
Numerous soluble and membrane enzymes that mediate the tricarboxylic acid (TCA) cycle were simultaneously monitored for their activity and expression by Blue Native Polyacrylamide Electrophoresis (BN-PAGE). These enzymes were characterized by the specific staining obtained following the precipitation of formazan upon incubation of the gel with their respective substrates. NAD+-dependent isocitrate dehydrogenase (NAD-ICDH), α-ketoglurarate dehydrogenase (KGDH), malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) were identified on the same gel. Fumarase (FUM) and aconitase (ACN) were detected through the formation of their products malate and isocitrate, respectively. Purple bands were observed when the gels were incubated with either MDH and iodonitrotetrazolium (INT) or ICDH and INT. Two dimensional (2D) BN-PAGE and/or 2D SDS-PAGE enabled the quantitation and purification of these enzymes. These results demonstrate that BN-PAGE can be adapted to measure the activity and relative expression of various TCA cycle enzymes and can also be utilized to study the fate of these enzymes in various biological systems.
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How to cite this article:

Ranji Singh , Robert Hamel , Daniel Chenier , Ryan Mailloux , Robin Beriault , Jeffrey Middaugh and Vasu D. Appanna , 2006. Simultaneous Monitoring of Activities of Numerous Tricarboxylic Acid Cycle Enzymes by Blue Native Polyacrylamide Gel Electrophoresis. Asian Journal of Biochemistry, 1: 297-306.

DOI: 10.3923/ajb.2006.297.306

URL: https://scialert.net/abstract/?doi=ajb.2006.297.306

 
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