Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis
Article first published online: 9 NOV 2005
The Plant Journal
Volume 44, Issue 5, pages 893–901, December 2005
How to Cite
Sabar, M., Balk, J. and Leaver, C. J. (2005), Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis. The Plant Journal, 44: 893–901. doi: 10.1111/j.1365-313X.2005.02577.x
- Issue published online: 9 NOV 2005
- Article first published online: 9 NOV 2005
- Received 21 June 2005; revised 30 August 2005; accepted 1 September 2005.
- histochemical staining;
- respiratory chain;
- plant electron transfer complexes
Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F1F0-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.