Abstract
Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217(2):220–230
Pitceathly RD, Rahman S, Wedatilake Y, Polke JM, Cirak S, Foley AR, Sailer A, Hurles ME, Stalker J, Hargreaves I, Woodward CE, Sweeney MG, Muntoni F, Houlden H, Taanman JW, Hanna MG, U.K. Consortium (2013) NDUFA4 mutations underlie dysfunction of a cytochrome c oxidase subunit linked to human neurological disease. Cell Rep 3(6):1795–1805
Van Coster R, Smet J, George E, De Meirleir L, Seneca S, Van Hove J, Sebire G, Verhelst H, De Bleecker J, Van Vlem B, Verloo P, Leroy J (2001) Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr Res 50(5):658–665
Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32(4):529–539
Perez-Perez R, Lobo-Jarne T, Milenkovic D, Mourier A, Bratic A, Garcia-Bartolome A, Fernandez-Vizarra E, Cadenas S, Delmiro A, Garcia-Consuegra I, Arenas J, Martin MA, Larsson NG, Ugalde C (2016) COX7A2L Is a mitochondrial complex III binding protein that stabilizes the III2+IV supercomplex without affecting respirasome formation. Cell Rep 16(9):2387–2398
Enriquez JA (2016) Supramolecular organization of respiratory complexes. Annu Rev Physiol 78:533–561
Lapuente-Brun E, Moreno-Loshuertos R, Acin-Perez R, Latorre-Pellicer A, Colas C, Balsa E, Perales-Clemente E, Quiros PM, Calvo E, Rodriguez-Hernandez MA, Navas P, Cruz R, Carracedo A, Lopez-Otin C, Perez-Martos A, Fernandez-Silva P, Fernandez-Vizarra E, Enriquez JA (2013) Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Science 340(6140):1567–1570
Shoubridge EA (2012) Supersizing the mitochondrial respiratory chain. Cell Metab 15(3):271–272
Fernandez-Vizarra E, Ferrin G, Perez-Martos A, Fernandez-Silva P, Zeviani M, Enriquez JA (2010) Isolation of mitochondria for biogenetical studies: an update. Mitochondrion 10(3):253–262
Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428
Acknowledgments
This work was supported by GOSH/Sparks Charity grant “From the biology of EPG5 to the pathophysiology of Vici syndrome” to M.R.D.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Singh, K., Duchen, M.R. (2022). Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE. In: Tomar, N. (eds) Mitochondria. Methods in Molecular Biology, vol 2497. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2309-1_25
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2309-1_25
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2308-4
Online ISBN: 978-1-0716-2309-1
eBook Packages: Springer Protocols