Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed by isolating mitochondrial proteins from mutant and control cell lines, as detailed previously (58 (link),59 (link)). Samples containing 30 μg of total cellular proteins were separated on 3–12% Bis–Tris Native PAGE gel. The total OXPHOS Human WB Antibody Cocktail was applied as primary antibodies for this experiment. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as secondary antibodies and protein signals were detected using the ECL system (CWBIO).
Protocol detail
Blue Native PAGE Analysis of OXPHOS
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Anti igg
Antibodies
Antibody cocktail
Bis tris
Cell lines
Cellular
Goat
Human
Mitochondrial proteins
Mouse
Native page
Peroxidase
Protein
Rabbit
Corresponding Organization :
Other organizations : Zhejiang University, Children's Hospital of Zhejiang University
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Variable analysis
independent variables
- Mutant cell lines
- Control cell lines
- Mitochondrial protein expression levels
- OXPHOS protein levels
- Total cellular protein amount (30 μg) used for separation
- 3–12% Bis–Tris Native PAGE gel used for separation
- OXPHOS Human WB Antibody Cocktail used as primary antibodies
- Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG used as secondary antibodies
- ECL system (CWBIO) used for protein signal detection
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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