Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE

doi:10.1007/978-1-0716-2309-1_25

. 2022:2497:339-348.

doi: 10.1007/978-1-0716-2309-1_25.

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE

Kritarth Singh¹, Michael R Duchen²

Affiliations

¹ Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, UCL, London, UK.
² Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, UCL, London, UK. m.duchen@ucl.ac.uk.

PMID: 35771456
DOI: 10.1007/978-1-0716-2309-1_25

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE

Kritarth Singh et al. Methods Mol Biol. 2022.

. 2022:2497:339-348.

doi: 10.1007/978-1-0716-2309-1_25.

Authors

Kritarth Singh¹, Michael R Duchen²

Affiliations

¹ Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, UCL, London, UK.
² Department of Cell and Developmental Biology and Consortium for Mitochondrial Research, UCL, London, UK. m.duchen@ucl.ac.uk.

PMID: 35771456
DOI: 10.1007/978-1-0716-2309-1_25

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.

Keywords: In-gel activity; Mitochondria; Oxidative phosphorylation; Primary fibroblasts; Supercomplex.

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References

1. 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 - DOI
1. 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 - DOI
1. 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 - DOI
1. Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32(4):529–539 - DOI
1. 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 - DOI

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[1] 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 - DOI

[2] 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 - DOI

[3] 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 - DOI

[4] 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 - DOI

[5] 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 - DOI

[6] 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 - DOI

[7] Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32(4):529–539 - DOI

[8] Acin-Perez R, Fernandez-Silva P, Peleato ML, Perez-Martos A, Enriquez JA (2008) Respiratory active mitochondrial supercomplexes. Mol Cell 32(4):529–539 - DOI

[9] 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 - DOI

[10] 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 - DOI

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Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE

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Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE

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