TY - JOUR
T1 - Resolving mitochondrial protein complexes using nongradient blue native polyacrylamide gel electrophoresis
AU - Yan, Liang Jun
AU - Forster, Michael J.
N1 - Funding Information:
This work was supported by award PO1AG022550 from the National Institute on Aging. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Aging or the National Institutes of Health. The authors thank Drake Zhang at ProtTech for his assistance in mass spectrometry peptide sequencing.
PY - 2009/6/15
Y1 - 2009/6/15
N2 - Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomics analysis of high-molecular-weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this article, we present an alternative BN-PAGE method that uses highly porous, nongradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I to V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be detected simultaneously on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, nongradient gel electrophoretic approach that should be useful in functional proteomics studies.
AB - Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomics analysis of high-molecular-weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this article, we present an alternative BN-PAGE method that uses highly porous, nongradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I to V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be detected simultaneously on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, nongradient gel electrophoretic approach that should be useful in functional proteomics studies.
KW - Blue native polyacrylamide gel electrophoresis
KW - Dihydrolipoamide dehydrogenase
KW - Mitochondria
KW - Protein complexes
UR - http://www.scopus.com/inward/record.url?scp=67349219973&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2009.03.043
DO - 10.1016/j.ab.2009.03.043
M3 - Article
C2 - 19348780
AN - SCOPUS:67349219973
VL - 389
SP - 143
EP - 149
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -