Isolation and Analysis of Mitochondrial Complexes

Mitochondria were isolated using Dounce or Balch homogenizers, followed by standard differential centrifugation^{13 (link),50 (link),70 (link)}. Experimental procedure and antibodies for NBGE were used as described previously^{57 (link),71 (link)}. In brief, digitonin-solubilised mitochondria were separated on NativePAGE Novex Bis-Tris 3–12% gradient gels. After electrophoresis, the gels were incubated in transfer buffer containing 0.1% SDS for 10 min and proteins were transferred to PVDF membranes probed with specific antibodies (all diluted 1:500, except for VDAC1 and HSP60 diluted 1:1000) against complex I (NDUFA9, ab14713, Abcam; or NDUFB8, ab110242, Abcam), CII (SDHA, 14715, Abcam), CIII (Core2, ab14745, Abcam), CIV (COXVa, ab110262, Abcam) and CV (ATP5A, ab14748, Abcam; or ATP5B, HPA001520, Sigma Aldrich), and VDAC1 (ab15895, Abcam) or HSP60 (12165S, Cell Signaling) as the loading control.

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Bezawork-Geleta A., Wen H., Dong L., Yan B., Vider J., Boukalova S., Krobova L., Vanova K., Zobalova R., Sobol M., Hozak P., Novais S.M., Caisova V., Abaffy P., Naraine R., Pang Y., Zaw T., Zhang P., Sindelka R., Kubista M., Zuryn S., Molloy M.P., Berridge M.V., Pacak K., Rohlena J., Park S, & Neuzil J. (2018). Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints. Nature Communications, 9, 2221.

Publication 2018

ab14713 ab110242 ab14745 ab110262 ab14748 HPA001520 ab15895

Antibodies Atp5b Bis tris Buffer Complex i Digitonin Electrophoresis Gels Membranes Mitochondria Proteins Pvdf Vdac1

Corresponding Organization : Griffith University

Other organizations : Shenzhen University, Czech Academy of Sciences, Institute of Biotechnology, Czech Academy of Sciences, Institute of Molecular Genetics, Czech Academy of Sciences, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Macquarie University, Park Centre for Mental Health, University of Queensland, Malaghan Institute of Medical Research, Seoul National University

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Variable analysis

independent variables

Isolation method (Dounce or Balch homogenizers)
Digitonin-solubilization of mitochondria

dependent variables

Protein separation on NativePAGE Novex Bis-Tris 3–12% gradient gels
Protein transfer to PVDF membranes
Protein detection using specific antibodies

control variables

Standard differential centrifugation for mitochondria isolation
Experimental procedure and antibodies for NBGE as described previously

positive controls

VDAC1 (ab15895, Abcam)
HSP60 (12165S, Cell Signaling)

negative controls

Not explicitly mentioned

Annotations

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